Respiratory syncytial viruses and methods of use

Abstract

Disclosed herein are respiratory syncytial virus vaccines and methods of immunization for delivering respiratory syncytial virus vaccines in a subject.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 501,497, filed May 11, 2023, and U.S. Provisional Application No. 63 / 627,541, filed January 31, 2024, the contents of which are incorporated herein by reference in their entirety.

[0002] CRADA Statement This invention was created in the course of a collaborative research and development agreement with the National Institutes of Health, U.S. Department of Health and Human Services. The U.S. government holds certain rights to this invention. Technical Field

[0003] This article discloses information about respiratory syncytial virus (RSV) vaccines and immunization methods for using RSV vaccines in pediatric subjects.

[0004] sequence list This application includes a sequence list that has been electronically submitted in XML format, and which is incorporated herein by reference in its entirety. The XML copy was created on April 18, 2024, named 01121-0051-00PCT-ST26.xml, and has a size of 42,875 bytes. Background of the Invention RSV is the leading cause of severe acute lower respiratory tract disease (LRI) in infants and children worldwide, and the most common cause of severe pneumonia requiring hospitalization in children. Globally, RSV is estimated to have caused approximately 33 million LRI cases and approximately 118,000 deaths in children under 5 years of age in 2015. It is estimated that in low- and middle-income countries, more than 80% of all RSV-related LRIs (RSV-LRI) and more than 50% of RSV-related deaths occur in infants ≥ 6 months of age.

[0006] Live attenuated intranasal RSV vaccines are an attractive option for pediatric immunization because they mimic mild natural infection and induce durable cellular, humoral, local, and systemic immunity. Several trials of live attenuated RSV candidate vaccines have demonstrated that these vaccines do not cause the vaccine-associated enhanced RSV disease observed in children receiving formalin-inactivated RSV.

[0007] Advances have been made in understanding RSV gene function and in using reverse genetics systems to engineer rationally designed attenuated RSV strains, including strains attenuated by deletion of the NS2 gene, such as the RSV ΔNS2 / Δ1313 / I1314L candidate vaccine presented in this paper. RSV NS2 is a virus-encoded type I and type III interferon antagonist that interferes with interferon induction and signal transduction. In chimpanzees, intranasal and intratracheal inoculation with a wild-type RSV-A2 recombinant strain lacking the NS2 gene resulted in reduced replication in the upper and lower respiratory tracts compared to wild-type (wt)-RSV, as well as significant resistance to subsequent stimulation by wild-type RSV. Deletion of the NS2 gene elicits an increased interferon response to RSV infection, as demonstrated against bovine RSV lacking either NS1 or NS2 in calves. NS2 also acts as a pathogenic factor, promoting epithelial cell shedding in vitro and in hamster models, and its deletion may potentially reduce small airway obstruction, thereby improving the safety profile of such candidate vaccines. The deletion of the NS2 gene may be beneficial for vaccine safety, and the additional deletion of codon 1313 in the polymerase (L) gene (which also confers slight temperature sensitivity (shutdown temperature of 38°C to 39°C [100.4°F to 102.2°F])) may be beneficial for increased safety. The deletion of codon 1313 is further stabilized genetically and phenotypically by replacing isoleucine (I) with leucine (L) at codon 1314. Summary of the Invention

[0008] In particular, the following implementation schemes are provided here: This article discloses a method for immunizing pediatric subjects against respiratory syncytial virus (RSV) infection, which involves administering a dose of nebulized RSV vaccine to the pediatric subjects, the RSV vaccine containing an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L.

[0009] This article discloses a method for immunizing pediatric subjects against respiratory syncytial virus (RSV) infection, which involves administering a dose of RSV vaccine containing an effective amount of live attenuated RSV ∆NS2 / ∆1313 / I1314L to the pediatric subjects using an intranasal nebulizer delivery device.

[0010] This article discloses a method for preventing RSV infection in pediatric subjects or reducing the likelihood of RSV infection in pediatric subjects or preventing or reducing at least one symptom of RSV infection in pediatric subjects, the method comprising administering a dose of an RSV vaccine to the pediatric subject, the RSV vaccine containing an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L.

[0011] This article discloses a method for immunizing pediatric subjects against respiratory syncytial virus (RSV) infection, comprising administering to the pediatric subject a dose of an nebulized RSV vaccine containing an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L, wherein the effective amount of RSV comprises approximately 5 to approximately 9 log [unclear text - likely a typo]. 10 Plaque-forming units (PFU) / dose, optionally the intermediate dose is about 5.6 log 10 PFU / dosage.

[0012] This article discloses a method for immunizing pediatric subjects against respiratory syncytial virus (RSV) infection. The method involves administering a dose of an nebulized RSV vaccine to the pediatric subject. The RSV vaccine contains an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L, wherein the effective amount of RSV comprises approximately 5.4 log [amount missing] per dose. 10 PFU, approximately 5.6 log per dose 10 PFU, approximately 6.2 log per dose 10 PFU, approximately 6.4 log per dose. 10 PFU or approximately 7.0 log per dose. 10 PFU.

[0013] This article discloses a method for immunizing pediatric subjects against respiratory syncytial virus (RSV) infection, comprising administering a dose of RSV vaccine to the pediatric subject, the RSV vaccine containing an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L, wherein the effective amount of RSV comprises approximately 5 to approximately 9 log 10 Plaque-forming units (PFU) / dose, optionally the intermediate dose is about 5.6 log 10 PFU / dosage.

[0014] This article discloses a method for immunizing pediatric subjects against respiratory syncytial virus (RSV) infection. The method involves administering a dose of an RSV vaccine to the pediatric subject, the RSV vaccine containing an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L, wherein the effective amount of RSV comprises approximately 5.4 log per dose. 10 PFU, approximately 5.6 log per dose 10 Plaque-forming units (PFU), approximately 6.2 log per dose. 10 PFU, approximately 6.4 log per dose. 10 PFU or approximately 7.0 log per dose. 10 PFU.

[0015] In some implementations, the effective amount of RSV comprises approximately 5 log 10PFU / dose approximately 9 log 10 PFU / dose, optionally approximately 5.4 log 10 PFU / dose, approximately 5.6 log 10 PFU / dose, approximately 6.2 log 10 PFU / dose, approximately 6.4 log 10 PFU / dose or approximately 7.0 log 10 PFU / dose. In some embodiments, the RSV vaccine is delivered intranasally, with approximately 1 / 2 of the dose delivered to each nostril of the pediatric subject. In some embodiments, the RSV vaccine is delivered in doses of approximately 0.2 mL, with approximately 0.1 mL delivered to each nostril of the pediatric subject. In some embodiments, the RSV vaccine is delivered sequentially or simultaneously to each nostril of the pediatric subject.

[0016] In some embodiments, the method includes delivering a second dose of the RSV vaccine. In some embodiments, the second dose contains approximately 5.4 log... 10 PFU. In some implementations, the second dose contains approximately 5.6 log... 10 PFU. In some implementations, the second dose contains approximately 6.2 log... 10 PFU. In some implementations, the second dose contains approximately 6.4 log... 10 PFU. In some implementations, the second dose contains approximately 7.0 log... 10 PFU. In some implementations, the second dose contains approximately 5 log... 10 PFU to approximately 9 log 10 PFU. In some embodiments, the second dose is administered approximately 40-50, 45-55, 55-60, 52-60, or 60-65 days after the initial dose. In some embodiments, the second dose is administered at least 56 days after the initial dose. In some embodiments, the second dose is delivered intranasally, with approximately ½ the dose delivered to each nostril of the pediatric subject. In some embodiments, the second dose of the RSV vaccine is delivered in approximately 0.2 mL, with approximately 0.1 mL delivered to each nostril of the pediatric subject. In some embodiments, the RSV vaccine is delivered sequentially or simultaneously to each nostril of the pediatric subject.

[0017] In some implementations, the pediatric subjects are approximately 6 months to approximately 22 months old. In some implementations, the pediatric subjects are approximately 6 months to approximately 18 months old. In some implementations, the pediatric subjects are at least 6 months old. In some implementations, the pediatric subjects are full-term babies. In some implementations, the pediatric subjects are preterm babies.

[0018] In some embodiments, an intranasal nebulizer is used to administer a dose of RSV vaccine to a pediatric subject. In some embodiments, the intranasal nebulizer includes a nozzle for nebulizing the RSV vaccine for administration to the pediatric subject. In some embodiments, the intranasal nebulizer includes a cylinder, a plunger, and a dose dispenser. In some embodiments, the method includes advancing the plunger a first distance within the cylinder to deliver approximately half of the dose of the RSV vaccine to the first nostril of the pediatric subject. In some embodiments, the method includes removing the dose dispenser from the plunger. In some embodiments, the method includes advancing the plunger a second distance within the cylinder to deliver approximately half of the dose to the second nostril of the pediatric subject.

[0019] In some implementations, the intranasal nebulizer delivers droplets with an average droplet size of approximately 10–120 μm. v50 In some implementations, the average droplet size D delivered to each nostril of the pediatric subject is... v50 The droplet size is approximately 10 µm to 120 µm, approximately 30 µm to 120 µm, approximately 50 µm to 110 µm, approximately 70 µm to 110 µm, or approximately 80 µm to 110 µm. In some embodiments, the intranasal nebulizer delivers an average droplet size Dv50 of at least 30 µm, at least 50 µm, at least 70 µm, at least 80 µm, at least 110 µm, or at least 120 µm. In some embodiments, the average injection weight delivered to each nostril of a pediatric subject is approximately 30 mg to approximately 200 mg, approximately 50 mg to approximately 175 mg, approximately 70 mg to approximately 160 mg, approximately 80 mg to approximately 150 mg, 95 mg to approximately 135 mg, approximately 100 mg to approximately 130 mg, approximately 100 mg to approximately 130 mg, or between approximately 105 mg and approximately 130 mg. In some implementations, the average injection volume delivered to each nostril of a pediatric subject is about 85 µL to about 120 µL, about 90 µL to about 115 µL, or about 95 µL to about 115 µL.

[0020] In some embodiments, the codon at position 1313 of the serine residue encoding the L protein in the attenuated live RSV is deleted, resulting in a deletion of an amino acid in the L protein (∆1313). In some embodiments, the substitution of the amino acid residue of leucine for isoleucine at position 1314 in the attenuated live RSV causes a genetically stable mutation in the L gene (I1314L). In some embodiments, the attenuated live RSV comprises a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome containing the deletion of a codon encoding serine at position 1313 or the corresponding position of the L protein; a mutation at amino acid sequence residue 1314 or the corresponding position of the L protein, wherein the mutation at amino acid sequence residue 1314 of the L protein is a leucine-to-isoleucine amino acid substitution, wherein leucine is encoded by a codon as shown in CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

[0021] In some embodiments, the at least one symptom is selected from the development or onset of an upper respiratory tract RSV infection, the development or onset of a lower respiratory tract RSV infection, the development or onset of otitis media, the progression of an upper respiratory tract RSV infection to a lower respiratory tract RSV infection, or the progression to otitis media. In some embodiments, the at least one symptom is selected from wheezing, shortness of breath, or a combination thereof.

[0022] This article discloses the use of an intranasal nebulizer delivery device for administering a dose of RSV vaccine to pediatric subjects, the RSV vaccine containing an effective amount of RSV ∆NS2 / ∆1313 / I1314L.

[0023] This article discloses the use of an RSV vaccine in the manufacture of a medicine for preventing or reducing the likelihood of RSV infection in pediatric subjects, the RSV vaccine containing an effective amount of RSV ∆NS2 / ∆1313 / I1314L.

[0024] In some implementations, the effective amount of RSV comprises approximately 5 log per dose. 10 PFU to approximately 9 log 10 PFU. In some implementations, the effective amount of RSV comprises approximately 5.4 log. 10 PFU, approximately 5.6 log 10 PFU, approximately 6.2 log 10 PFU, approximately 6.4 log 10 PFU or approximately 7.0 log10 PFU. In some embodiments, the RSV vaccine is delivered intranasally, with approximately 1 / 2 of the dose delivered to each nostril of the pediatric subject. In some embodiments, the RSV vaccine is delivered in doses of approximately 0.2 mL, with approximately 0.1 mL delivered to each nostril of the pediatric subject. In some embodiments, the RSV vaccine is delivered sequentially or simultaneously to each nostril of the pediatric subject.

[0025] In some embodiments, this use includes delivering a second dose of the RSV vaccine. In some embodiments, the second dose contains approximately 5.4 log... 10 PFU. In some implementations, the second dose contains approximately 5.6 log... 10 PFU. In some implementations, the second dose contains approximately 6.2 log... 10 PFU. In some implementations, the second dose contains approximately 6.4 log... 10 PFU. In some implementations, the second dose contains approximately 7.0 log... 10 PFU. In some embodiments, the second dose contains approximately 5 log10 PFU to approximately 9 log10 PFU. 10 PFU. In some embodiments, the second dose is administered approximately 40-50, 45-55, 55-60, 52-60, or 60-65 days after the initial dose. In some embodiments, the second dose is administered at least 56 days after the initial dose. In some embodiments, the second dose is delivered intranasally, with approximately ½ the dose delivered to each nostril of the pediatric subject. In some embodiments, the second dose of the RSV vaccine is delivered in approximately 0.2 mL, with approximately 0.1 mL delivered to each nostril of the pediatric subject. In some embodiments, the RSV vaccine is delivered sequentially or simultaneously to each nostril of the pediatric subject.

[0026] In some implementations, the pediatric subjects are approximately 6 months to approximately 22 months old. In some implementations, the pediatric subjects are approximately 6 months to approximately 18 months old. In some implementations, the pediatric subjects are at least 6 months old. In some implementations, the pediatric subjects are full-term births. In some implementations, the pediatric subjects are preterm births.

[0027] In some embodiments, an intranasal nebulizer is used to administer a dose of RSV vaccine to a pediatric subject. In some embodiments, the intranasal nebulizer includes a nozzle for nebulizing the RSV vaccine for administration to the pediatric subject. In some embodiments, the intranasal nebulizer includes a cylinder, a plunger, and a dose dispenser. In some embodiments, the method includes advancing the plunger a first distance within the cylinder to deliver approximately half of the dose of the RSV vaccine to the first nostril of the pediatric subject. In some embodiments, the method includes removing the dose dispenser from the plunger. In some embodiments, the method includes advancing the plunger a second distance within the cylinder to deliver approximately half of the dose to the second nostril of the pediatric subject.

[0028] In some implementations, the intranasal nebulizer delivers droplets with an average droplet size of approximately 10–120 μm. v50 In some implementations, the average droplet size D delivered to each nostril of the pediatric subject is... v50 The droplet size is approximately 10 µm to 120 µm, approximately 30 µm to 120 µm, approximately 50 µm to 110 µm, approximately 70 µm to 110 µm, or approximately 80 µm to 110 µm. In some embodiments, the intranasal nebulizer delivers an average droplet size Dv50 of at least 30 µm, at least 50 µm, at least 70 µm, at least 80 µm, at least 110 µm, or at least 120 µm. In some embodiments, the average injection weight delivered to each nostril of a pediatric subject is approximately 30 mg to approximately 200 mg, approximately 50 mg to approximately 175 mg, approximately 70 mg to approximately 160 mg, approximately 80 mg to approximately 150 mg, 95 mg to approximately 135 mg, approximately 100 mg to approximately 130 mg, approximately 100 mg to approximately 130 mg, or between approximately 105 mg and approximately 130 mg. In some implementations, the average injection volume delivered to each nostril of a pediatric subject is about 85 µL to about 120 µL, about 90 µL to about 115 µL, or about 95 µL to about 115 µL.

[0029] In some embodiments, the codon at position 1313 of the serine residue encoding the L protein in the attenuated live RSV is deleted, resulting in a deletion of an amino acid in the L protein (∆1313). In some embodiments, the substitution of the amino acid residue of leucine for isoleucine at position 1314 in the attenuated live RSV causes a genetically stable mutation in the L gene (I1314L). In some embodiments, the attenuated live RSV comprises a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome containing the deletion of a codon encoding serine at position 1313 or the corresponding position of the L protein; a mutation at amino acid sequence residue 1314 or the corresponding position of the L protein, wherein the mutation at amino acid sequence residue 1314 of the L protein is a leucine-to-isoleucine amino acid substitution, wherein leucine is encoded by a codon as shown in CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

[0030] This article discloses a kit containing a dose of RSV vaccine and an intranasal nebulization delivery device for administering the RSV vaccine to pediatric subjects. The RSV vaccine contains an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L.

[0031] In some implementations, the effective amount of RSV comprises approximately 5 log per dose. 10 PFU to approximately 9 log 10 PFU. In some implementations, the effective amount of RSV comprises approximately 5.4 log. 10 PFU, and / or approximately 5.6 log 10 PFU, and / or approximately 6.2 log 10 PFU, and / or approximately 6.4 log 10 PFU, and / or approximately 7.0 log 10 PFU. In some embodiments, the kit also contains a second dose of RSV vaccine. In some embodiments, the second dose contains an effective amount of the RSV, the effective amount of the RSV comprising approximately 5 log... 10 PFU to approximately 9 log 10 PFU / Second Dose. In some embodiments, the second dose contains an effective amount of RSV, comprising approximately 5.4 log... 10 PFU and / or approximately 5.6 log 10 PFU and / or approximately 6.2 log 10PFU and / or approximately 6.4 log 10 PFU and / or approximately 7.0 log 10 PFU / Second Dose. In some embodiments, the first and / or second dose comprises a volume of approximately 0.2 mL.

[0032] In some embodiments, the intranasal nebulization delivery device includes a nozzle to nebulize the RSV vaccine. In some embodiments, the intranasal nebulization delivery device includes a cylinder, a plunger, and a dose dispenser. In some embodiments, the intranasal nebulization delivery device delivers droplets with an average droplet size of approximately 10-120 μm. v50 In some embodiments, the intranasal nebulizer delivers an average droplet size Dv50 of at least 30 µm, at least 50 µm, at least 70 µm, at least 80 µm, at least 110 µm, or at least 120 µm. In some embodiments, the intranasal nebulizer delivers an average injection weight of half a dose between about 30 mg and about 200 mg, about 50 mg and about 175 mg, about 70 mg and about 160 mg, about 80 mg and about 150 mg, 95 mg and about 135 mg, about 100 mg and about 130 mg, about 100 mg and about 130 mg, or about 105 mg and about 130 mg. In some embodiments, the intranasal nebulizer delivers an average injection volume of half a dose between about 85 µL and about 120 µL, about 90 µL and about 115 µL, or about 95 µL and about 115 µL.

[0033] This article discloses a recombinant infectious respiratory syncytial virus comprising a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), M2-1 protein nonstructural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH); and a genome or antigenome comprising the deletion of a codon encoding serine at position 1313 or the corresponding position of the L protein; a mutation at amino acid sequence residue 1314 or the corresponding position of the L protein, wherein the mutation at amino acid sequence residue 1314 of the L protein is a leucine substitution for isoleucine, wherein leucine is encoded by a codon as shown in CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A). Brief description of the attached diagram Figure 1 illustrates the Phase I / II study scheme according to the implementation scheme of this disclosure.

[0035] Figure 2 illustrates an intranasal atomization delivery device according to an embodiment of the present disclosure.

[0036] Figure 3 illustrates a dose dispenser according to an embodiment of this disclosure.

[0037] Figure 4 illustrates an intranasal nebulizer delivery device with a dose dispenser according to an embodiment of the present disclosure.

[0038] Figure 5 illustrates the genomic structure of RSV ΔNS2 / Δ1313 / I1314L. Detailed Implementation

[0039] This article describes methods for immunizing subjects against respiratory syncytial virus (RSV) infection. These methods may include administering RSV vaccine to subjects using an intranasal nebulizer delivery device.

[0040] Unless otherwise defined, all technical terms, symbols, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, for clarity and / or ease of reference, terms with commonly understood meanings are defined herein, and the inclusion of these definitions herein should not be construed as representing a difference from the commonly understood meaning in the art. The techniques and procedures described or referenced herein are generally well-known to those skilled in the art and are typically employed using conventional methods, such as, for example, the widely adopted molecular cloning method described below, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. As applicable, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions, unless otherwise stated.

[0041] I. Definition Unless otherwise stated, the following terms and phrases used herein shall have the following meanings: As used in this article, "attenuated live virus" refers to a viable virus that exhibits reduced, weakened, or no clinical symptoms when administered to subjects. A "attenuated live vaccine" contains attenuated live virus.

[0042] As used herein, a “vector” or “vector-based” virus or vaccine contains a virus modified to express one or more heterologous antigens. In some cases, a “vector” or “vector-based” vaccine is also a live attenuated vaccine, wherein the vaccine, when administered to a subject, exhibits reduced, attenuated, or no clinical symptoms.

[0043] As used herein, “prevention” means preventing or avoiding disease manifestations, delaying the onset of one or more symptoms of a particular disease, disorder, or condition (e.g., RSV infection), and / or reducing the frequency and / or severity of such one or more symptoms. In some implementations, prevention is assessed on a population basis such that if a statistically significant reduction in the development, frequency, and / or intensity of one or more symptoms of a particular disease, disorder, or condition is observed in a population susceptible to such a disease, disorder, or condition, the agent is considered to “prevent” the disease, disorder, or condition.

[0044] As used herein, the terms "vaccination" or "vaccinate" refer to the administration of a composition intended to produce an immune response, for example, against a pathogen. Vaccination may be administered before, during, and / or after exposure to a pathogen and / or the appearance of one or more symptoms, and in some embodiments, shortly before, during, and / or after exposure to the pathogen. In some embodiments, vaccination comprises multiple administrations of a vaccination composition at appropriate intervals.

[0045] As used herein, “RSV ΔNS2 / Δ1313 / I1314L” refers to RSV ΔNS2 / Δ1313 / I1314L (NIH) or RSV ΔNS2 / Δ1313 / I1314L (Sanofi). Each of ΔNS2 / Δ1313 / I1314L (NIH) and RSV ΔNS2 / Δ1313 / I1314L (Sanofi) contains an attenuated live RSV with (i) a 523-nucleotide (nt) deletion in the NS2 gene (ΔNS2), (ii) an amino acid deletion in the L protein, and (iii) a genetically stable mutation in the L gene (see Figure 5). The attenuated live RSV ∆NS2 / ∆1313 / I1314L (Sanofi) also contains a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A) in the non-coding region.

[0046] As used herein, “RSV ΔNS2 / Δ1313 / I1314L vaccine” refers to either “RSV ΔNS2 / Δ1313 / I1314L (NIH) vaccine” or “RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine”. The RSV ΔNS2 / Δ1313 / I1314L (NIH) vaccine contains an effective amount of RSV ΔNS2 / Δ1313 / I1314L (NIH). The RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine contains an effective amount of RSV ΔNS2 / Δ1313 / I1314L (Sanofi).

[0047] As used herein, the term "immune response" refers to the response of cells of the immune system (such as B cells, T cells, dendritic cells, macrophages, or polymorphonuclear cells) to a stimulus (such as an antigen or vaccine). An immune response can include any cell in the body involved in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate and / or adaptive immune responses. As used herein, a "protective immune response" refers to an immune response that protects a subject from infection (e.g., prevents infection or the occurrence of infection-related diseases). Methods for measuring an immune response include measuring, for example, the proliferation and / or activity of lymphocytes (such as B or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, etc. An "antibody response" is an immune response that produces antibodies.

[0048] As used in this article, "immunity" refers to actions that induce an immune response in a subject or protect the subject from infection.

[0049] As used herein, “adjuvant” refers to a substance or medium that nonspecifically enhances an immune response against an antigen. Adjuvants may include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) to which antigens are adsorbed; water-in-oil or oil-in-water emulsions of antigen solutions emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Sometimes, killed mycobacteria (e.g., Freund's complete adjuvant) are included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) may also be used as adjuvants (e.g., see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants may also include biomolecules such as Toll-like receptor (TLR) agonists and co-stimulatory molecules. Exemplary biological adjuvants include, but are not limited to, IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, or combinations thereof.

[0050] As used in this article, “liquid” is given its traditional meaning, and “frozen liquid” is a solid liquid that is distinct from a liquid.

[0051] "Formulation" refers to a composition containing an active pharmaceutical ingredient or biological component and one or more other components. The term "formulation" is used interchangeably herein with the terms "pharmaceutical composition," "vaccine composition," and "vaccine formulation." Depending on the circumstances, other components that may be included include pharmaceutically acceptable excipients, additives, diluents, buffers, sugars, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), chelating agents, surfactants, polyols, fillers, stabilizers, lyophilization protectants, solubilizers, emulsifiers, salts, adjuvants, tension enhancers (such as alkali metal halides (e.g., sodium chloride or potassium chloride), mannitol, or sorbitol), delivery media, and antimicrobial preservatives.

[0052] As used herein, “treat,” “treating,” and “treatment” mean any administration or application of a therapeutic agent to a disease or disorder in a subject, and include suppressing the disease, preventing its progression, alleviating one or more symptoms of the disease, curing the disease, or preventing the recurrence of one or more symptoms of the disease. In some cases, treatment includes reducing or improving the progression, severity, and / or duration of upper and / or lower respiratory tract RSV infection, otitis media, or related symptoms or respiratory conditions such as wheezing, shortness of breath, or a combination thereof.

[0053] As used herein, a “therapeutic effective amount” or “effective amount” is an amount of composition or its active component sufficient to provide a beneficial effect or otherwise reduce harmful or non-beneficial events in an individual to whom the composition is administered. As used herein, reference to “therapeutic effective amount” or “effective amount” means a dose that produces one or more desired or desirable (e.g., beneficial) effects to which it is administered, administered once or more over a given period of time. The exact dose will depend on the purpose of treatment and will be determined using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0054] The term “about” or “approximately” is used herein to mean approximately, roughly, roughly, or within a range. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the value. Generally, the term “about” can modify a value to be higher or lower (higher or lower) by variance (e.g., 10%, up or down). In some embodiments, the term indicates a deviation from the indicated value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, “about” indicates a deviation from the indicated value of ±10%. In some embodiments, “about” indicates a deviation from the indicated value of ±5%. In some embodiments, “about” indicates a deviation from the indicated value of ±4%. In some embodiments, “about” indicates a deviation from the indicated value of ±3%. In some embodiments, “about” indicates a deviation from the indicated value of ±2%. In some embodiments, "about" indicates a deviation of ±1% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.9% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.8% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.7% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.6% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.5% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.4% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.3% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.2% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.1% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.05% from the indicated value. In some embodiments, "about" indicates a deviation of ±0.01% from the indicated value. All ranges proposed herein are intended to include both the lower and upper limits of the ranges.

[0055] The term "pediatric subject" refers to a subject who is 21 years of age or younger at the time of immunization. Pediatric subgroups are further characterized as: (i) neonates – from birth to 28 days after birth; (ii) infants and toddlers – 29 days to under 2 years of age; (iii) children – 2 years to under 12 years of age; and (iv) adolescents – 12 years to 21 years of age. In some respects, pediatric subjects may be 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year of age or younger. In some respects, pediatric subjects may be at least 6 months old. In some respects, pediatric subjects may be 6 months to 18 years of age. In some respects, pediatric subjects may be 6 months to 10 years of age. In some respects, pediatric subjects may be 4 months to 4 years of age. In some respects, the age of pediatric subjects may be 6 to 18 months. In some respects, the age of pediatric subjects may be 6 to 22 months. In some respects, the age of pediatric subjects may be 6 months to less than 24 months. The age of pediatric subjects may also include other ranges. In some respects, full-term pediatric subjects as defined herein are those born at ≥37 weeks of gestation. In some respects, pediatric subjects are preterm, as defined herein as those born at 28 to 36 weeks of gestation.

[0056] Reference will now be made in detail to certain embodiments, examples of which are shown in the accompanying drawings. While embodiments have been described in conjunction with the illustrated embodiments, it should be understood that they are not intended to limit this disclosure to those embodiments. Rather, this disclosure is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the disclosure as defined in the appended claims and include the embodiments.

[0057] Before describing the teachings of this disclosure in detail, it should be understood that this disclosure is not limited to specific compositions or method steps and is therefore subject to variation. It should be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used in this specification and the appended claims include plural indicators. Thus, for example, a reference to “conjugate” includes multiple conjugates and a reference to “cell” includes multiple cells, etc.

[0058] Numerical ranges include numbers within a defined range. Taking into account significant figures and measurement-related errors, measured and measurable values ​​should be understood as approximate values. Furthermore, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be restrictive. It should be understood that the foregoing general and detailed descriptions are exemplary and interpretive only, and not intended to limit the content of the teachings.

[0059] Unless otherwise specified in the specification, embodiments in the specification that list "comprising" various components are also considered to be "consisting of the listed components" or "substantially composed of the listed components"; embodiments in the specification that list "consisting of various components" are also considered to "comprising the listed components" or "substantially composed of the listed components"; and embodiments in the specification that list "substantially composed of various components" are also considered to "consisting of the listed components" or "including the listed components" (this interchangeability does not apply to the use of these terms in the claims). The term "or" is used in an inclusive sense, i.e., equivalent to "and / or," unless the context clearly indicates otherwise.

[0060] The section headings used herein are for organizational purposes only and should not be construed in any way as limiting the intended subject matter. If any material incorporated by reference contradicts any terminology defined herein or any other express content herein, this specification shall prevail. While the teachings herein are described in conjunction with various embodiments, it is not intended to limit the teachings herein to those embodiments. Rather, as those skilled in the art will understand, the teachings herein include various alternatives, modifications, and equivalents.

[0061] II. Exemplary Methods and Applications In some embodiments, a method is provided to immunize a subject against respiratory syncytial virus (RSV) infection, which includes administering an nebulized dose of RSV vaccine. In some embodiments, a method is provided to immunize a subject against RSV infection, which includes administering a dose of RSV vaccine using an intranasal nebulizer delivery device.

[0062] In some embodiments, methods for immunizing a subject against RSV infection include administering an nebulized dose of an RSV vaccine containing live attenuated RSV. In some embodiments, methods for immunizing a subject against RSV infection include administering a dose of an RSV vaccine containing live attenuated RSV using an intranasal nebulizer delivery device. In some embodiments, a method for immunizing a subject against RSV infection is provided, comprising administering an nebulized dose of an RSV vaccine, optionally using an intranasal nebulizer delivery device, the RSV vaccine comprising: an effective amount of attenuated live RSV having (i) a 523-nucleotide (nt) deletion in the NS2 gene (∆NS2), (ii) an amino acid deletion in the L protein, and (iii) a genetically stable mutation in the L gene (RSV∆NS2 / ∆1313 / I1314L (NIH)); or further comprising RSV with a nucleotide modification at position 14456 of reference SEQ ID NO: 1, the modification being in a non-coding region representing a change from thymine (T) to adenine (A) (RSV ∆NS2 / ∆1313 / I1314L (Sanofi)). In some embodiments, the codon encoding the serine at position 1313 of the L protein is deleted, resulting in an amino acid deletion in the L protein (∆1313). In some implementations, the substitution of leucine for isoleucine at position 1314 causes a genetically stable mutation (I1314L) in the L gene.

[0063] In some embodiments, a method is provided for immunizing pediatric subjects against RSV infection, the method comprising administering an nebulized dose of RSV vaccine. In some embodiments, a method is provided for immunizing pediatric subjects against RSV infection, the method comprising administering a dose of RSV vaccine using an intranasal nebulizer. In some embodiments, the pediatric subject may be at least 6 months old. In some embodiments, the pediatric subject may be 6 months to 18 months old. In some embodiments, the pediatric subject may be 6 months to 22 months old. In various embodiments, the pediatric subject may be 6 months to less than 24 months old. In some embodiments, the pediatric subject is born at full term. In some embodiments, the pediatric subject is preterm.

[0064] In some embodiments, a method is provided for preventing RSV infection in a subject, reducing the likelihood of RSV infection in a subject, or preventing or reducing at least one symptom of RSV infection in a subject, the method comprising administering a nebulized dose of RSV vaccine. In some embodiments, a method is provided for preventing RSV infection in a subject, reducing the likelihood of RSV infection in a subject, or preventing or reducing at least one symptom of RSV infection in a subject, the method comprising administering a dose of RSV vaccine using an intranasal nebulizer delivery device.

[0065] In some embodiments, methods for preventing RSV infection in a subject, reducing the likelihood of RSV infection in a subject, or preventing or reducing at least one symptom of RSV infection in a subject include administering a nebulized dose of an RSV vaccine containing attenuated live RSV. In some embodiments, methods for preventing RSV infection in a subject, reducing the likelihood of RSV infection in a subject, or preventing or reducing at least one symptom of RSV infection in a subject include administering a nebulized dose of an RSV vaccine, optionally using a nebulized delivery device, the RSV vaccine containing an effective amount of RSV ∆NS2 / ∆1313 / I1314L. In some embodiments, the codon encoding serine at position 1313 of the L protein is deleted, resulting in a deletion of the amino acid (∆1313) in the L protein. In some implementations, the substitution of leucine for isoleucine at position 1314 causes a genetically stable mutation (I1314L) in the L gene.

[0066] In some embodiments, a method is provided for preventing RSV infection in a subject or reducing the likelihood of RSV infection in a subject, or for preventing or reducing at least one symptom of RSV infection in a pediatric subject, the method comprising administering a nebulized dose of RSV vaccine. In some embodiments, a method is provided for preventing RSV infection in a subject or reducing the likelihood of RSV infection in a subject, or for preventing or reducing at least one symptom of RSV infection in a pediatric subject, the method comprising administering a dose of RSV vaccine using an intranasal nebulizer. In some embodiments, the pediatric subject may be at least 6 months old. In some embodiments, the pediatric subject may be 6 months to 18 months old. In some embodiments, the pediatric subject may be 6 months to 22 months old. In some embodiments, the pediatric subject may be 6 months to less than 24 months old. In some embodiments, the pediatric subject is full-term. In some embodiments, the pediatric subject is preterm.

[0067] In some embodiments, the pediatric subject is 6 months to 21 years old. In some embodiments, the pediatric subject is 21 years old or younger. In some embodiments, the pediatric subject is 10 to 21 years old. In some embodiments, the pediatric subject is 5 to 10 years old. In some embodiments, the pediatric subject is 12 months to 5 years old. In some embodiments, the pediatric subject may be at least 6 months old. In some embodiments, the pediatric subject is 6 months to 36 months old. In some embodiments, the pediatric subject is 6 months to less than 24 months old. In some embodiments, the pediatric subject is 6 months to 18 months old. In some embodiments, the pediatric subject is 6 months to 22 months old. In some implementations, the age of pediatric subjects is 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36 months. In some implementations, the age of pediatric subjects is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 years. In some implementations, pediatric subjects are full-term infants. In some implementations, the pediatric subjects were preterm infants. In some implementations, the pediatric subjects had a prior RSV infection. In some implementations, the pediatric subjects did not have an RSV infection.

[0068] In some implementations, when used in conjunction with any of the methods described herein, the effective amount of RSV comprises approximately 5.4 log. 10 Plaque-forming units (PFU) / dose. In some embodiments, when used with any of the methods described herein, the effective amount of RSV comprises approximately 5.6 log. 10 Plaque-forming units (PFU) / dose. In some embodiments, used in conjunction with any of the methods described herein, the effective amount of RSV comprises approximately 6.2 log. 10 Plaque-forming units (PFU) / dose. In some embodiments, when used with any of the methods described herein, the effective amount of RSV comprises approximately 6.4 log. 10 Plaque-forming units (PFU) / dose. In some embodiments, when used with any of the methods described herein, the effective amount of RSV comprises approximately 7.0 log. 10Plaque-forming units (PFU) / dose. In some embodiments, when used in conjunction with any of the methods described herein, the effective amount of RSV comprises approximately 5.4 log. 10 PFU up to 7.0log 10 PFU. In some implementations, when used with any of the methods described herein, the effective amount of RSV comprises approximately 5.6 log. 10 PFU to 6.2 log 10 PFU / dose. In some embodiments, when used with any of the methods described herein, the effective amount of RSV comprises approximately 5.6 log. 10 PFU down to 6.4 log 10 PFU / dose. In some embodiments used with any of the methods described herein, the effective amount of RSV comprises approximately 5 log [units missing]. 10 PFU, 5.1 log 10 PFU, 5.2log 10 PFU, 5.3 log 10 PFU, 5.4 log 10 PFU, 5.5 log 10 PFU, 5.6 log 10 PFU, 5.7 log 10 PFU, 5.8log 10 PFU, 5.9 log 10 PFU, 6 log 10 PFU, 6.1 log 10 PFU, 6.2 log 10 PFU, 6.3 log 10 PFU, 6.4log 10 PFU, 6.5 log 10 PFU, 6.6 log 10 PFU, 6.7 log 10 PFU, 6.8 log 10 PFU, 6.9 log 10 PFU, 7log 10 PFU, 7.1 log 10 PFU, 7.2 log 10 PFU, 7.3 log 10 PFU, 7.4 log 10 PFU, 7.5 log 10 PFU, 7.6log 10 PFU, 7.7 log 10 PFU, 7.8 log 10PFU, 7.9 log 10 PFU, 8 log 10 PFU, 8.1 log 10 PFU, 8.2log 10 PFU, 8.3 log 10 PFU, 8.4 log 10 PFU, 8.5 log 10 PFU, 8.6 log 10 PFU, 8.7 log 10 PFU, 8.8log 10 PFU, 8.9 log 10 PFU and / or 9 log 10 PFU / dose. In some embodiments, when used with any of the methods described herein, the effective amount of RSV comprises approximately 5 log [units missing]. 10 PFU to 9 log 10 PFU / dose. In some embodiments, when used with any of the methods described herein, the effective amount of RSV comprises approximately 5 log [units missing]. 10、 6 log 10 7log 10 8 log 10 or 9 logs 10 PFU / dose. In some embodiments, when used with any of the methods described herein, the effective amount of RSV comprises approximately 5 log [units missing]. 10 PFU to approximately 9 log 10 PFU / dosage.

[0069] In some embodiments, the RSV vaccine is delivered intranasally in a nebulized dose. In some embodiments, the RSV vaccine is delivered intranasally using an intranasal nebulizer. In some embodiments, approximately half the dose is delivered to each nostril. In some embodiments, the RSV vaccine is delivered intranasally so that the entire dose is delivered to one nostril. In some embodiments, the RSV vaccine is delivered intranasally, wherein half the dose is delivered to one nostril, and another half the dose is delivered sequentially or simultaneously to the same nostril. In some embodiments, the RSV vaccine is delivered intranasally, delivering unequal doses to one or both nostrils. In some embodiments, the RSV vaccine is delivered intranasally, wherein ¾ of the dose is delivered to one nostril, and ¼ of the dose is delivered to the other nostril or the same nostril. In some implementations, the RSV vaccine is delivered intranasally, with 1 / 10, 1 / 9, 1 / 8, 1 / 7, 1 / 6, 1 / 5, ¼, 1 / 3, ½, or the whole dose delivered to one nostril, and an additional 9 / 10, 8 / 9, 7 / 8, 5 / 6, 4 / 5, ¾, 2 / 3, or ½ dose delivered to the other nostril or the same nostril.

[0070] In some embodiments, the RSV vaccine is delivered intranasally as a liquid formulation. In some embodiments, the RSV vaccine dose is delivered intranasally in a volume of about 0.2 mL. In some embodiments, the 0.2 mL dose is delivered intranasally, with about 0.1 mL delivered to each nostril. In some embodiments, an intranasal nebulizer is used to deliver the RSV vaccine dose intranasally in volumes of about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, or 1.0 mL. As described above, the dose can be evenly distributed between the two nostrils, substantially evenly distributed between the two nostrils, unevenly distributed between the two nostrils, or delivered entirely to one nostril in one or more deliveries. In some embodiments used in conjunction with any of the methods described herein, an intranasal nebulizer is used to deliver the RSV vaccine intranasally. In some embodiments, the intranasal nebulizer may include RSV vaccine in a pre-filled cartridge.

[0071] In some embodiments, a method for immunizing a subject against respiratory syncytial virus (RSV) infection is provided, which includes administering a first dose of RSV vaccine and administering a second dose of RSV vaccine. In some embodiments, the method for immunizing a subject against RSV infection may include administering one or more doses using an intranasal nebulizer delivery device.

[0072] In some implementations, a method for immunizing a subject against RSV infection is provided, comprising: administering a first dose of an RSV vaccine containing attenuated live RSV, optionally using an intranasal nebulizer; and administering a second dose of the RSV vaccine, optionally using an intranasal nebulizer.

[0073] In some embodiments, a method is provided for preventing RSV infection in a subject or reducing the likelihood of RSV infection in a subject, or preventing or reducing at least one symptom of RSV infection in a subject, the method comprising administering a first dose of RSV vaccine and administering a second dose of RSV vaccine. In some embodiments, a method is provided for preventing RSV infection in a subject or reducing the likelihood of RSV infection in a subject, or preventing or reducing at least one symptom of RSV infection in a subject, wherein one or more doses of RSV vaccine are administered using an intranasal nebulizer delivery device.

[0074] In some implementations, methods for preventing RSV infection in a subject or reducing the likelihood of RSV infection in a subject, or preventing or reducing at least one symptom of RSV infection in a subject, include: administering a dose of an RSV vaccine containing attenuated live RSV, optionally using an intranasal nebulizer; and administering a second dose of the RSV vaccine, optionally using an intranasal nebulizer.

[0075] In some implementations, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5.4 log per dose. 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5.6 log per dose. 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 6.2 log per dose. 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 6.4 log per dose. 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 7.0 log per dose. 10 PFU. In some implementations, when used with any of the methods described herein, the effective amount of RSV comprises approximately 5.4 log. 10 PFU up to 7.0 log 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5.4 log. 10PFU up to 7.0 log 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5.6 log per dose. 10 PFU to 6.2 log 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5.4 log per dose. 10 PFU. In some implementations, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5.6 log per dose. 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 6.2 log per dose. 10 PFU. In some implementations, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 6.4 log per dose. 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 7.0 log per dose. 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5.2 log per dose. 10 PFU up to 7.0 log 10 PFU. In some embodiments, when used with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5.6 log per dose. 10 PFU down to 6.4 log 10 PFU. In some embodiments used in conjunction with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5 log [missing information]. 10 PFU, 5.1 log 10 PFU, 5.2log 10 PFU, 5.3 log 10 PFU, 5.4 log 10 PFU, 5.5 log 10 PFU, 5.6 log 10 PFU, 5.7 log 10 PFU, 5.8log 10 PFU, 5.9 log 10 PFU, 6 log 10 PFU, 6.1 log 10 PFU, 6.2 log 10PFU, 6.3 log 10 PFU, 6.4log 10 PFU, 6.5 log 10 PFU, 6.6 log 10 PFU, 6.7 log 10 PFU, 6.8 log 10 PFU, 6.9 log 10 PFU, 7log 10 PFU, 7.1 log 10 PFU, 7.2 log 10 PFU, 7.3 log 10 PFU, 7.4 log 10 PFU, 7.5 log 10 PFU, 7.6log 10 PFU, 7.7 log 10 PFU, 7.8 log 10 PFU, 7.9 log 10 PFU, 8 log 10 PFU, 8.1 log 10 PFU, 8.2log 10 PFU, 8.3 log 10 PFU, 8.4 log 10 PFU, 8.5 log 10 PFU, 8.6 log 10 PFU, 8.7 log 10 PFU, 8.8log 10 PFU, 8.9 log 10 PFU and / or 9 log 10 PFU / dose. In some embodiments, when used in conjunction with any of the methods described herein, the effective amount of RSV in the second dose comprises approximately 5 log per dose. 10 approximately 9 log 10 PFU. In some embodiments, the effective amount of RSV in the second dose comprises approximately 5 log per dose. 10 Approximately 5.4 log 10 Approximately 5.6 log 10 Approximately 6 logs 10 Approximately 6.2 log 10 Approximately 6.4 log 10 Approximately 7 logs 10 Approximately 8 logs 10 Or approximately 9 logs 10PFU. In some embodiments, the first and second doses of the RSV vaccine are the same. In some embodiments, the first and second doses of the administered RSV vaccine are different. In some embodiments, the effective amount of RSV in the first and second doses contains approximately 5.4 log [amount missing]. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first and second doses comprises approximately 5.6 log [units unspecified]. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first and second doses comprises approximately 6.2 log 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first and second doses comprises approximately 6.4 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first and second doses comprises approximately 7.0 log [amount missing]. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first and second doses comprises approximately 5 log [value missing]. 10 PFU / dose approximately 9 log 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 5.4 log [units unspecified]. 10 PFU / dose, and the second dose contains approximately 5.6 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 5.4 log [units unspecified]. 10 PFU / dose, and the second dose contains approximately 6.2 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 5.4 log. 10 PFU / dose, and the second dose contains approximately 6.4 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 5.4 log 10 PFU / dose, and the second dose contains approximately 6.4 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 5.4 log. 10 PFU / dose, and the second dose contains approximately 7.0 log. 10 PFU / dosage. In some implementations, the effective amount of RSV in the first dose comprises approximately 5.6 log. 10 PFU / dose, and the second dose contains approximately 5.2 log. 10 PFU / dosage. In some implementations, the effective amount of RSV in the first dose comprises approximately 5.6 log. 10PFU / dose, and the second dose contains approximately 6.2 log. 10 PFU / dosage. In some implementations, the effective amount of RSV in the first dose comprises approximately 5.6 log. 10 PFU / dose, and the second dose contains approximately 6.4 log. 10 PFU / dosage. In some implementations, the effective amount of RSV in the first dose comprises approximately 5.6 log. 10 PFU / dose, and the second dose contains approximately 7.0 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 6.2 log. 10 PFU / dose, and the second dose contains approximately 5.4 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 6.2 log. 10 PFU / dose, and the second dose contains approximately 5.6 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 6.2 log. 10 PFU / dose, and the second dose contains approximately 6.4 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 6.2 log. 10 PFU / dose, and the second dose contains approximately 7.0 log. 10 PFU / dosage. In some implementations, the effective amount of RSV in the first dose comprises approximately 6.4 log 10 PFU / dose, and the second dose contains approximately 5.4 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 6.4 log. 10 PFU / dose, and the second dose contains approximately 5.6 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 6.4 log. 10 PFU / dose, and the second dose contains approximately 6.2 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 6.4 log. 10 PFU / dose, and the second dose contains approximately 7.0 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 7.0 log. 10 PFU / dose, and the second dose contains approximately 5.4 log. 10PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 7.0 log. 10 PFU / dose, and the second dose contains approximately 5.6 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 7.0 log. 10 PFU / dose, and the second dose contains approximately 6.2 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 7.0 log. 10 PFU / dose, and the second dose contains approximately 6.4 log. 10 PFU / dosage. In some embodiments, the effective amount of RSV in the first dose comprises approximately 5 log [units missing]. 10 Approximately 5.4 log 10 Approximately 5.6 log 10 Approximately 6 logs 10、 Approximately 6.2 log 10 Approximately 6.4 log 10 Approximately 7 logs 10 Approximately 8 logs 10 Or approximately 9 logs 10 PFU / dose and the second dose contains approximately 5 log. 10 Approximately 5.2 log 10 Approximately 5.6 log 10 Approximately 6 logs 10 Approximately 6.2 log 10 Approximately 6.4 log 10 Approximately 7 logs 10 Approximately 8 logs 10 Or approximately 9 logs 10 PFU / dosage.

[0076] In some embodiments, a single or multiple doses of a live attenuated RSV vaccine may be administered to the subjects described herein. Multiple doses (e.g., 2, 3, 4, or more doses) may be used in primary immunization and / or booster immunization regimens. Multiple doses are typically administered at intervals of at least one week (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, etc.). In some embodiments, the RSV vaccine is administered in a single dose. In some embodiments, a second RSV vaccine is administered once or more, optionally at intervals of about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, or about 16 weeks, or other suitable intervals. In some embodiments, the second RSV vaccine is administered about 7 to about 9 weeks after the first dose. In some embodiments, the second dose is administered about 40-50, 45-55, 55-60, 52-60, or 60-65 days after the first dose. In some embodiments, the two doses are administered approximately 56 days (8 weeks) apart. In some implementations, the two doses are administered at an interval of at least 56 days. In some implementations, a single or multiple doses of the live attenuated RSV vaccine can be administered using an intranasal nebulizer.

[0077] In some embodiments, administration of the RSV vaccine reduces the incidence of laboratory-confirmed RSV. In some embodiments, administration of the RSV vaccine reduces at least one symptom of RSV infection in subjects, wherein the symptom is selected from the development or onset of upper and / or lower respiratory tract RSV infection, otitis media, or related respiratory symptoms, or the progression of upper respiratory tract RSV infection to lower respiratory tract RSV infection, or progression to otitis media or related respiratory symptoms. In some embodiments, administration of the RSV vaccine reduces at least one symptom of RSV infection, wherein the symptom is selected from wheezing, shortness of breath, or a combination thereof.

[0078] In some embodiments, a method is provided to immunize a subject against respiratory syncytial virus (RSV) infection, the method comprising administering a first dose of RSV vaccine using an intranasal nebulizer delivery device, wherein the intranasal nebulizer delivery device includes a spray nozzle for nebulizing the RSV vaccine. In some embodiments, a spray plume from the nozzle may be directed toward the top of the nasal passage into the nasal cavity. In some embodiments, a method is provided to prevent RSV infection in pediatric subjects or reduce the likelihood of RSV infection in pediatric subjects or to prevent or reduce at least one symptom of RSV infection in pediatric subjects, the method comprising administering a dose of RSV vaccine using an intranasal nebulizer delivery device, wherein the intranasal nebulizer delivery device includes a spray nozzle for nebulizing the RSV vaccine. In some embodiments, the intranasal nebulizer delivery device includes: a cylinder operatively connected to the spray nozzle, and a plunger movable within the cylinder to propel the RSV vaccine through the spray nozzle. In some embodiments, the intranasal nebulizer delivery device further includes a dose dispenser for dividing the dose of the RSV vaccine into two or more deliveries. In some embodiments, the dose dispenser may divide the dose of the RSV vaccine into approximately half the volume to be delivered to the subject. In some implementations, a dose dispenser is used to deliver half a dose to each nostril of the subject.

[0079] In some implementations, the intranasal nebulizer delivers droplets with an average droplet size of 10-120 μm. v50 In some embodiments, the intranasal nebulizer delivers droplets of average size D of approximately 10 µm to 120 µm, approximately 20 µm to 120 µm, approximately 30 µm to 120 µm, approximately 40 µm to 120 µm, approximately 50 µm to 110 µm, approximately 60 µm to 110 µm, approximately 70 µm to 110 µm, or approximately 80 µm to 110 µm. v50In some embodiments, the intranasal nebulizer delivers an average droplet size Dv50 of at least 100 µm, at least 110 µm, or at least 120 µm. In some embodiments, the intranasal nebulizer delivers an average droplet size Dv50 of at least 30 µm, at least 50 µm, at least 70 µm, at least 80 µm, or at least 110 µm. In some embodiments, the intranasal nebulizer delivers an average injection weight between about 30 mg and about 200 mg, about 50 mg and about 175 mg, about 70 mg and about 160 mg, about 80 mg and about 150 mg, 95 mg and about 135 mg, about 100 mg and about 130 mg, about 100 mg and about 130 mg, or about 105 mg and about 130 mg. In some embodiments, the intranasal nebulizer delivers an average injection volume between about 25 µL and about 200 µL, about 50 µL and about 175 µL, about 60 µL and about 150 µL, about 75 µL and about 130 µL, about 85 µL and about 120 µL, about 90 µL and about 115 µL, or about 95 µL and about 115 µL.

[0080] In some embodiments, the intranasal nebulizer delivery device described herein is provided for administering a dose of RSV vaccine to a subject, wherein the RSV vaccine comprises an effective amount of live attenuated RSV. In some embodiments, the use includes live attenuated RSV ΔNS2 / Δ1313 / I1314L.

[0081] In some embodiments, the use of an RSV vaccine in the manufacture of a medicament for preventing or reducing the likelihood of RSV viral infection in a subject is provided, said RSV vaccine comprising an effective amount of attenuated live RSV. In some embodiments, said use includes RSV ΔNS2 / Δ1313 / I1314L.

[0082] III. RSV vaccine In some implementations, RSV vaccines may include live attenuated vaccines. In one instance, an RSV vaccine described as “RSV ΔNS2 / Δ1313 / I1314L vaccine” is a live attenuated vaccine based on the deletion (ΔNS2) of the gene encoding the RSV interferon / apoptosis antagonist NS2 protein. The deletion of the NS2 gene attenuates the virus and enhances immunity. The RSV ΔNS2 / Δ1313 / I1314L vaccine further contains a genetically stable attenuated and temperature-sensitive mutation in the L protein (codon deletion Δ1313, and a missense mutation I1314L that prevents the deattenuation mutation that could otherwise occur at position 1314), and therefore RSV ΔNS2 / Δ1313 / I1314L is temperature-sensitive, with a shut-off temperature of 38°C to 39°C (100.4°F to 102.2°F) against viral replication.

[0083] The RSV ΔNS2 / Δ1313 / I1314L vaccine is a recombinant infectious respiratory syncytial virus comprising a large polymerase protein (L), a phosphoprotein (P), a nucleocapsid protein (N), an M2-1 protein, non-structural protein 1 (NS1), a glycoprotein (G), a fusion protein (F), a matrix protein (M), an M2-2 protein, and a small hydrophobic protein (SH), and having a genome or anti-genome with the following: deletion of the codon encoding the L protein at position 1313 or the corresponding serine residue; mutation of the L protein amino acid sequence residue 1314 or the corresponding residue, wherein the mutation of the L protein amino acid sequence residue 1314 is a leucine-to-isoleucine amino acid substitution (wherein leucine is encoded by a codon as shown in CTG); and deletion of the NS2 gene. The RSV ΔNS2 / Δ1313 / I1314L vaccine and the method for preparing the vaccine are described in WO 2013 / 154728 A1, which is incorporated herein by reference in its entirety for any purpose.

[0084] IV. Intranasal nebulizer delivery device A. Apparatus In some embodiments, an intranasal nebulization delivery device can be used to deliver the vaccine described herein. The intranasal nebulization delivery device can be any suitable device for nebulizing a live attenuated RSV vaccine. In some embodiments, the delivery device can provide an average droplet size Dv of approximately 10 μm to 120 μm. 50In some embodiments, the delivery device is suitable for delivery to pediatric subjects. In some embodiments, the delivery device is suitable for delivery to pediatric subjects aged 6 months to 18 months. In various embodiments, the delivery device is suitable for delivery to pediatric subjects aged 6 months to less than 24 months. In some embodiments, the delivery device is suitable for delivery to pediatric subjects at least 6 months old. In various embodiments, the delivery device may be configured to deliver two substantially equal doses, one dose per nostril. In some embodiments, the two doses are delivered sequentially or simultaneously.

[0085] By way of non-limiting example, a suitable intranasal nebulization delivery device 10 is described. As shown in FIG2, the intranasal nebulization delivery device 10 may include a cartridge 4 for receiving and delivering a vaccine to a subject. The delivery device 10 may also include a Luer lock 3 adapted to receive a vial access cannula 2 and / or a nebulizer 6. The delivery device may include a cap 1 to cover the vial access cannula 2. The delivery device 10 may also include a plunger 5 sized and shaped to fit within the cartridge 4. The cartridge 4 may also include a marking for identifying the volume within the cartridge 4 of the fluid volume used to deliver the vaccine, as shown in FIG4. As a non-limiting example, the marking may be a 0.05 mL marking, a 0.1 mL marking, a 0.2 mL marking, or other markings, depending on the dose to be delivered and whether it is administered into one or both nostrils. In some embodiments, the cartridge 4 may be a 1 mL syringe. As a non-limiting example, the syringe may be a 1 mL Luer lock disposable plastic syringe, such as B. Braun Omnifix®-F. In some embodiments, the cartridge 4 may be a smaller or larger volume syringe. The vial access cannula 2 can be used to retrieve the vaccine from a vial at a medical center for delivery to a subject. In some embodiments, the vial access cannula 2 may be a semi-blunt-tipped or pointed-tipped filling needle. By way of non-limiting example, the semi-blunt-tipped filling needle may be 18G x 40mm (B. Braun Sterican® MIX). Once the cartridge 4 is filled with the appropriate vaccine dose, the vial access cannula 2 can be removed, and the nebulizer 6 can be connected to the cartridge 4 via a Luer lock 3. The nebulizer 6 can be infused before administering the vaccine dose intranasally. The nebulizer 6 may be generally conical with a wide base that tapers towards the tip for insertion into the nostril, allowing a spray plume to exit from the nebulizer 6, as described below. In some embodiments, the cartridge 4 may be pre-filled with the vaccine dose to be delivered.

[0086] In some embodiments, the intranasal delivery device 10 may have a typical droplet size of about 10 µm to 120 µm and a system dead zone of about 0.1 mL to 0.2 mL. In some embodiments, the system dead zone may be about 0.15 mL. The nebulizer 6 may have a tip diameter of about 3.5 mm to 5 mm to facilitate delivery into the nasal cavity. In some embodiments, the tip diameter may be about 4 mm to 4.5 mm, and may be about 4.3 mm. As a non-limiting example, the Teleflex MAD130 Nasal... TM Device system or Teleflex Vaxinator TM The (VAX300) device can be used to deliver the vaccine intranasally to the subject. In some implementations, the nebulizer 6 may be an Aptar, LuerVax® device.

[0087] B. Dosage dispenser In some embodiments, the dose dispenser 12 (see Figures 3 and 4) may be provided together with the intranasal nebulizer delivery device 10 of Figure 2. The dose dispenser 12 can be used to dispense the vaccine dose provided in the intranasal nebulizer delivery device 10. In some embodiments, the dose dispenser 12 can be used to divide the vaccine dose into two substantially equal half-volume vaccine doses (e.g., a first half-dose and a second half-dose), delivering independently half a volume for each nostril or to both half volumes of the same nostril. Other dose dispensing is also possible and depends on the height of the dose dispenser. As shown in Figures 3 and 4, the dose dispenser 12 may include a first portion 14 having a height corresponding to the portion of the dose to be delivered to the first nostril. The first portion 14 may also include a notch 16 whose size and shape are determined to correspond to the size and shape of the plunger 5. The dose dispenser 12 may also include a second portion 18 including two protrusions 20 that can be used to widen the opening 22 in the first portion 14 so that the notch 16 can be positioned above the plunger 5 of the delivery device 10. The two protrusions 20 can be released to close the first portion 14 above the plunger 5, thereby controlling the volume of the first portion dose delivered to the first nostril. The dose dispenser 12 can be removed from the plunger 5 to deliver a second portion dose to the subject's second nostril. As a non-limiting example, the dose dispenser (such as dose dispenser 12) can be used to deliver approximately 0.01 mL / nostril, 0.02 mL / nostril, 0.05 mL / nostril, 0.075 mL / nostril, 0.1 mL / nostril, 0.2 mL / nostril, 0.3 mL / nostril, 0.4 mL / nostril, 0.5 mL / nostril, 0.6 mL / nostril, 0.7 mL / nostril, 0.8 mL / nostril, 0.9 mL / nostril, or 1.0 mL / nostril. Other volumes may also be used. In some embodiments, the dose dispenser 12 can divide the dose into two approximately equal half-dose portions.

[0088] C. Dosage delivery The vaccine can be delivered to a subject intranasally using the delivery device 10. In some embodiments, the dose can be delivered in approximately half to each nostril, a larger volume to the first nostril and a smaller volume to the second nostril, or the same or different volumes can be administered twice to the same nostril. The dose delivery described herein is for delivering approximately half the dose to each nostril, but is not limited thereto. The full dose to be delivered to the subject can be loaded into the delivery device 10 by removing the vaccine from a container (e.g., a vial; not shown) containing one or more doses of vaccine using the vial access cannula 2. Once the full dose has been loaded into the cylinder 4 of the delivery device 10, the vial access cannula 2 can be removed from the Luer lock 3, and the nebulizer 6 can be coupled to or connected to the Luer lock 3. Furthermore, the nebulizer 6 can be infused, and the dose dispenser 12 can be connected to the plunger 5 as described above. With the nebulizer 6 inserted into the first nostril, the plunger 5 can be advanced a first distance within the cylinder 4 to force the first half dose through the nebulizer 6, thereby nebulizing the vaccine to form a spray plume. The spray plume may comprise droplet fines ranging in size from approximately 10 μm to approximately 120 μm. The remote region of the nebulizer 6 may be generally conical. The spray plume may be generally directed toward the top of the nasal passage and enter the nasal cavity through the nasal flap, including the nasal turbinate regions (including the inferior, middle, and superior nasal turbinate regions). The plunger 5 may be advanced a first distance within the tube 4 until the tip of the plunger 5 contacts the dose dispenser 12 and stops advancing to deliver a first half-dose to the first nostril.

[0089] After the first half-dose is delivered to the first nostril, the dose dispenser 12 can be removed from the plunger 5. The nebulizer 6 can then be inserted into the second nostril, and the plunger 5 can be advanced a second distance within the cylinder 4 to deliver the second half-dose to the second nostril. The second half-dose is completed when the plunger 5 contacts the end of the cylinder 4.

[0090] Example The following examples are provided to illustrate certain disclosed implementations and should not be construed in any way as limiting the scope of this disclosure.

[0091] Example 1. A safety, immunogenicity, infectivity, and dosage investigation of an experimental attenuated live RSV vaccine in infants and young children. Execution Summary Research Products Recombinant attenuated live respiratory syncytial virus (RSV) ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine.

[0092] Active substances The attenuated live RSV has (i) a 523 nucleotide (nt) deletion in the NS2 gene (ΔNS2), (ii) an amino acid deletion in the L protein (Δ1313; deletion of S1313), and (iii) a genetically stable mutation in the L gene (I1314L) (RSVΔNS2 / Δ1313 / I1314L (Sanofi)).

[0093] Research Location A multicenter, multinational study that included approximately 30 sites in the United States, approximately 2 sites in Canada, approximately 6 sites in Latin America (Argentina, Chile, and Honduras), and approximately 2 sites in South Africa.

[0094] Phase I / II study A phase I / II randomized, observer-blinded, placebo-controlled, multicenter dose-finding study was conducted in the United States, Canada, Latin America (Argentina, Chile, and Honduras), and South Africa to evaluate the safety, immunogenicity, infectivity, and vaccine shedding of a single or two doses of the live attenuated RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine delivered via intranasal nebulizer in infants and young children aged 6 to 18 months (cohorts 1 to 4). Figure 1 shows the study protocol of the example, where the dose can be any of the doses described in Example 1.

[0095] Vaccination: A total of 300 infants and toddlers aged 6 to 18 months were sequentially enrolled into one of four cohorts and, within each cohort, randomly assigned to receive intranasal administration of their designated study product using an intranasal nebulizer delivery device as follows: Cohort 1 – 40 infants and toddlers received a single administration of 1:1 ratio RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 5.6 log 10 PFU (low dose) or placebo (same formulation buffer as RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine).

[0096] Cohort 2 – 40 infants and toddlers received two doses at a 1:1 ratio of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 5.6 log 10 PFU (low dose) or placebo.

[0097] Cohort 3 – 40 infants and toddlers received a single administration of 1:1 ratio RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 6.2 log 10 PFU (high dose) or placebo.

[0098] Cohort 4 – 180 infants and toddlers received two doses of a 1:1:1 RSV ΔNS2 / Δ1313 / I1314L (Sanofi) at a concentration of 5.6 log. 10 PFU (low dose), RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 6.2 log 10 PFU (high dose) or placebo.

[0099] [Table 1]: Vaccination cohort Detailed content Research rationale Prior to the RSV ΔNS2 / Δ1313 / I1314L vaccine presented in this paper, three RSV vaccine viruses with NS2 gene deletions (rA2cpΔNS2, rA2cp248 / 404ΔNS2, and rA2cp530 / 1009ΔNS2) had been evaluated in clinical studies (Wright et al., 2006, J. Infect Dis., 193(4):573-81). The rA2cpΔNS2 candidate was over-attenuated in adults but under-attenuated for use in children and adolescents, while both rA2cp248 / 404ΔNS2 and rA2cp530 / 1009ΔNS2 were over-attenuated and under-immunized in seronegative children. Based on these results, RSV ΔNS2 / Δ1313 / I1314L (NIH) was developed. In a recent phase Ia study sponsored by the National Institutes of Health (NIH) in 6- to 24-month-old RSV serologically negative children (n = 20) (Karron et al., 2020, JInfect Dis.; 222(1):82-91), in 10 6 At PFU doses, RSV ∆NS2 / ∆1313 / I1314L (National Institutes of Health) is safe, has good infectivity (100% of vaccinated subjects were infected) and immunogenicity (80% of subjects had a >4-fold increase in neutralizing antibody titer), and is well-suited for a strong recall response to wild-type (wt) RSV infection in post-season RSV surveillance.

[0100] Research Objectives Main objectives To assess the safety profile of each dose of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) after each and any administration using the intranasal nebulizer delivery device in all infants and young children, regardless of baseline serum status.

[0101] In subjects who had not been infected with RSV, the RSVA serum neutralizing antibody response to the study product was characterized in each vaccine group after vaccination 1 (D56) for cohorts 1, 2, 3 and 4 and after vaccination 2 (D84) for cohorts 2 and 4.

[0102] Secondary objectives The amount of vaccine virus shed by each participant in cohorts 1, 2, 3, and 4 at D7 and for cohorts 2 and 4 at D63 was quantified by baseline serological status, which was measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR).

[0103] The proportion of vaccinated infants and young children in each vaccine cohort who contracted the vaccine virus after vaccination 1 (D56) for cohorts 1, 2, 3, and 4, and after vaccination 2 (D84) for cohorts 2 and 4, was determined by baseline serological status. (Infection was defined as a ≥ 4-fold increase in the titer of vaccine and / or RSV A serum antibody titer or RSV serum anti-F IgG antibody titer detected by polymerase chain reaction (PCR) in nasal swab samples.) In subjects who had experienced RSV, the RSV A serum neutralizing antibody response to the study product was characterized in each vaccine group after vaccination 1 (D56) for cohorts 1, 2, 3 and 4 and after vaccination 2 (D84) for cohorts 2 and 4.

[0104] Baseline serological status was used to characterize the RSV serum anti-FIgG antibody response to the study product in each vaccine group after vaccination 1 (D56) for cohorts 1, 2, 3, and 4, and after vaccination 2 (D84) for cohorts 2 and 4.

[0105] Baseline serological status was used to characterize the RSV serum antibody response (RSV A neutralization and anti-RSV F IgG) to the study product in each vaccine group at least 5 months after the RSV surveillance season or after the last vaccine administration.

[0106] Exploratory goals Exploratory safety goals The safety profile of RSV was assessed using baseline serum status for each dose and after any administration.

[0107] Exploratory immunogenicity targets: • The RSVA serum neutralizing antibody response to the study product was characterized by baseline serological status for each vaccine group after vaccination 1 (D56) for cohorts 1, 2, 3 and 4 and after vaccination 2 (D84) for cohorts 2 and 4 (converted to IU / mL values).

[0108] • Characterize the RSV A serum neutralizing antibody response to the study product by baseline serum status, which is converted to IU / mL values ​​after the RSV season or at least 5 months after the last vaccine administration.

[0109] • Baseline serological status was used to characterize the RSV B serological neutralizing antibody response to the study product in each vaccine group after vaccination 1 (D56) for cohorts 1, 2, 3, and 4, and after vaccination 2 (D84) for cohorts 2 and 4.

[0110] • Characterize the RSV B serum neutralizing antibody response to the study product in each vaccine group (converted to IU / mL values) by antibody serological status for cohorts 1, 2, 3 and 4 after vaccination 1 (D56) and for cohorts 2 and 4 after vaccination 2 (D84).

[0111] • Characterize the RSV B serum neutralizing antibody response to the study product in each vaccine group by antibody serological status after the RSV season or at least 5 months after the last vaccine administration.

[0112] • Characterize the RSV B serum neutralizing antibody response to the study product by antibody serological status, which is converted to IU / mL values ​​after the RSV season or at least 5 months after the last vaccine administration.

[0113] • Baseline serological status was used to characterize the serum anti-G protein central conserved region (Gcc) IgG antibody response to the study product in each vaccine group after vaccination 1 (D56) for cohorts 1, 2, 3 and 4, and after vaccination 2 (D84) for cohorts 2 and 4.

[0114] • Characterize the serum anti-Gcc IgG antibody response to the study product in each vaccine group at least 5 months after the RSV season or after the last vaccine administration by antibody serological status.

[0115] • Baseline serological status characterizes RSV serum anti-F IgA antibody response in cohorts 1, 2, 3, and 4 after vaccination 1 (D56) and in cohorts 2 and 4 after vaccination 2 (D84).

[0116] • Characterize RSV serum anti-F IgA antibody response by baseline serum status after RSV season or at least 5 months after the last vaccine administration.

[0117] Exploratory efficacy goals Describe the frequency and severity of RSV-related medically required acute respiratory illness (RSV MAARI) and RSV-related medically required acute lower respiratory illness (RSV MAALRI) in all infants and young children in each vaccine group during RSV season or at least 5 months after the last vaccination.

[0118] Primary safety endpoint: In all infants and young children, regardless of baseline serum status: • Any actively administered systemic adverse events reported within 30 minutes of each vaccination.

[0119] • Requested application site reactions and systemic reactions (i.e., pre-listed in the subject's DC / eDC and CRB) occur within 28 days after each and any vaccination (i.e., the acute phase).

[0120] • Any AE that is proactively reported (self-reported) occurs within 28 days of each and any vaccination.

[0121] • The occurrence of any AESI within 28 days after each vaccination.

[0122] • Any MAAE occurring within 28 days after each vaccination.

[0123] • Any SAEs occurring throughout the research process.

[0124] • Other safety endpoints will be documented or derived as described in the statistical analysis plan. Depending on the project, these may include: nature (preferred term in the MedDRA), time of onset, duration, number of days, intensity, relationship to vaccine, actions taken, whether the AE led to early termination of the study, severity, or outcome.

[0125] Primary immunogenic endpoint: RSV A serum neutralizing antibody titers in subjects who had not been infected with RSV at D56 for cohorts 1, 2, 3 and 4 and at D84 for cohorts 2 and 4.

[0126] For the sponsor's Phase I / II study, data will be analyzed based on baseline serological status. Baseline serological status will be determined retrospectively from serum samples collected from baseline (V01). Participants will be categorized as having experienced RSV or not having been infected with RSV based on the presence or absence of detected RSV serum anti-F IgA antibodies. This biomarker was chosen because it is produced only in response to RSV infection and does not transfer from mother to child across the placenta.

[0127] Research Plan Description of the overall research design and plan Research Design This is a phase I / II randomized, observer-blinded, placebo-controlled, multicenter dose-exploration study conducted in the United States, Canada, Latin America (Argentina, Chile, and Honduras), and South Africa. It was used to evaluate the safety, immunogenicity, infectivity, and vaccine shedding of a live attenuated RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine administered via intranasal nebulizer to infants and young children aged 6 to 18 months (cohorts 1 to 4) after one or two doses.

[0128] Vaccination: A total of 300 infants and toddlers aged 6 to 18 months were either sequentially enrolled or will be enrolled in one of four cohorts, and within each cohort will be either randomly assigned to receive or will be randomly assigned to receive intranasal administration of the designated study product using an intranasal nebulizer delivery device as follows: Cohort 1 – 40 infants and toddlers received or will receive a single dose at a 1:1 ratio of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 5.6 log 10 PFU (low dose) or placebo (same formulation buffer as RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine) Cohort 2 – 40 infants and toddlers who have received or will receive two doses at a 1:1 ratio of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 5.6 log 10 PFU (low dose) or placebo Cohort 3 – 40 infants and toddlers who have received or will receive a single dose, 1:1 ratio RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 6.2 log 10 PFU (high dose) or placebo Cohort 4 – 180 infants and toddlers received two doses of a 1:1:1 RSV ∆NS2 / ∆1313 / I1314L (Sanofi) 5.6 log 10 PFU (low dose), RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 6.2 log 10 PFU (high dose) or placebo.

[0129] Queue 1 (Northern Hemisphere): 1:1 randomized, placebo-controlled, single-dose RSV ΔNS2 / Δ1313 / I1314L (Sanofi) (5.6 log) 10 PFU was administered once on day (D) 0 (total 0.2 mL per administration), with n = 20 in each vaccine cohort. Enrollment was initiated at approximately 15 sites in the United States. Vaccine administration to enrolled subjects was completed at least 5 days prior to the start of the RSV season (the average 5-month RSV season in the Northern Hemisphere is November 1 to March 31). No further enrollment was attempted in subsequent cohorts because recruitment for cohort 1 did not reach the target of n = 40.

[0130] Queue 2 (Southern Hemisphere): 1:1 randomized, placebo-controlled, single-dose RSV ΔNS2 / Δ1313 / I1314L (Sanofi) (5.6 log) 10 PFU (Pulsed Fuel Activated) will be administered twice, on days 0 and 56 (total 0.2 mL each time), with n = 20 participants per vaccine cohort. Enrollment will begin as early as November / December 2020 at approximately four sites in Latin America (Argentina and Chile). The first and second doses of vaccine (on days 0 and 56, respectively) for enrolled participants will be completed at least 5 days prior to the start of the RSV season (the average 5-month RSV season in the Southern Hemisphere is from May 1 to September 30). If recruitment for cohort 1 does not reach the target of n = 40, no further attempts will be made to enroll more participants in subsequent cohorts.

[0131] Queue 3 (Northern Hemisphere): 1:1 randomized, placebo-controlled, single-dose RSV ΔNS2 / Δ1313 / I1314L (Sanofi) (6.2 log) 10 PFU was administered once on day 0 (total 0.2 mL per administration), with n = 20 in each vaccine cohort. Enrollment began in April 2021 at approximately 30 sites in the United States. Vaccination of enrolled subjects was completed by May 31, 2021. Because recruitment in cohort 3 did not reach the target of n = 40, no attempt was made to include more subjects receiving a single administration in cohort 4.

[0132] Queue 4 (Northern and Southern Hemispheres): 1:1:1 Randomized placebo-controlled two-dose RSV ΔNS2 / Δ1313 / I1314L (Sanofi) (5.6 log) 10 PFU or 6.2 log 10 PFU was administered twice, on days 0 and 56 (total 0.2 mL each time), with n = 60 in each vaccine group. Enrollment of subjects was initiated as early as June 2021 at approximately 30 sites in the United States, as early as May 2022 at approximately 2 sites in Canada, as early as November 2021 at approximately 2 sites in Chile, as early as April 2022 at approximately 2 sites in Honduras, and as early as June 2022 at approximately 2 sites in South Africa.

[0133] The first and second doses of the vaccine for enrolled subjects were administered at any time of year, including the winter RSV season, regardless of whether the normal RSV seasonality had resumed after interruptions caused by non-pharmacological interventions for COVID-19.

[0134] Blood sampling: All subjects selected for inclusion in cohorts 1, 2, 3, and 4 provided a blood sample upon enrollment (visit 01) for baseline RSV serum antibody testing.

[0135] All subjects in each vaccine group in cohorts 1, 2, 3, and 4 presented blood samples at day 56 (previously before vaccination 2 for cohorts 2 and 4) to measure post-vaccination serum antibody titers against RSV. All subjects in each vaccine group in cohorts 2 and 4 presented blood samples at day 84 (28 days post-vaccination 2) to measure post-vaccination serum antibody titers against RSV. All subjects in cohorts 1, 2, 3, and 4 presented blood samples one month after the RSV season or at least five months after the last vaccination to measure post-season RSV antibody titers, thus determining whether a four-fold or greater increase in RSV antibody titers had occurred during the RSV season, indicating that wild-type RSV infection was not detected during surveillance.

[0136] Nasal swab sample: For cohorts 1, 2, 3, and 4, nasal swab samples were collected from all enrolled subjects on day 7; and for cohorts 2 and 4, nasal swab samples were collected on day 63 for the following purposes: The vaccine virus shedding was quantified in D7 of cohorts 1, 2, 3 and 4, and in D63 of cohorts 2 and 4.

[0137] If subjects become ill at the same time point, respiratory pathogens are tested on the same nasal swab samples (D7 for cohorts 1, 2, 3 and 4, and D63 for cohorts 2 and 4).

[0138] Nasal swab samples were collected from subjects during illness visits and at any other time point specified in the study protocol 48 hours later (including surveillance requiring medical attention during RSV season) for the detection of RSV and respiratory pathogens.

[0139] Collection of security data: The acute phase of the first vaccination begins on day 0 and ends at midnight on day 28. The acute phase of the second vaccination begins on day 56 and ends at midnight on day 84. Any adverse reactions (AEs, AESIs, MAAEs, or SAEs) that begin within the acute phase (i.e., within 28 days of vaccination) but are diagnosed by a healthcare professional after 28 days are still considered to have occurred within the acute phase.

[0140] The acute-after phase for subjects receiving a single dose began at 12:01 a.m. on D29 and ended at midnight on D56. For subjects receiving two doses, the first acute-after phase began at 12:01 a.m. on D29 and ended at midnight on D56, unless the second dose was administered exactly on D56 immediately before the subject's first dose. The second acute-after phase for subjects receiving two doses began at 12:01 a.m. on D85 and ended at midnight on D112.

[0141] All subjects were observed for 30 minutes after each vaccine administration, and any actively administered systemic adverse events (AEs) occurring during this period were recorded in the case report book (CRB) as immediately administered systemic AEs.

[0142] All subjects were followed up for requested application site reactions and systemic reactions, proactive adverse events, adverse events of particular concern (AESI), and adverse events requiring medical attention (MAAE) within 28 days after each and any vaccination and from vaccination to the end of study participation in response to serious adverse events (SAE).

[0143] Parents / guardians / legally authorized representatives of the participants recorded information in a Diary Card (DC) / Electronic Diary Card (eDC) to capture responses to requests, proactive adverse events (AEs), AESIs, and MAAEs for Cohorts 1, 2, 3, and 4 from D0 to D28, and for Cohorts 2 and 4 from D56 to D84. Parents / guardians / legally authorized representatives reported responses, adverse events, and any symptoms suggestive of respiratory illness to the study site. Follow-up was conducted by telephone at the study site. The eDC also allowed for daily safety monitoring of study participants. In some cases, when the eDC was unavailable, paper diary cards were used for daily safety monitoring. Responses to all requests were graded using the Sponsor Intensity Scale, except for runny nose / nasal discharge and nasal congestion or obstruction graded using the Department of Acute Respiratory Diseases (DAIDS) scale. Adverse events of particular concern, except for wheezing, were graded using the DAIDS scale, with wheezing graded according to the Brighton Collaboration guidelines.

[0144] Based on the National Institutes of Health's (NIH) prior clinical experience with live attenuated RSV candidate vaccines and the sponsor's standard practice for the collection, analysis, and reporting of safety data, responses to the following predefined requests were evaluated for each vaccine administration during the acute phase: administration site reactions including runny nose and nasal congestion or nasal blockage / congestion, and systemic reactions including fever, vomiting, unusual crying, lethargy, loss of appetite, and irritability.

[0145] The following AESIs were assessed during the acute phase of the study: acute otitis media, upper respiratory tract infection (URI) (including pharyngitis and cough without lower respiratory tract infection (LRI)), and lower respiratory tract infection (LRI) (including wheezing, rales, tachypnea, acute wheezing, pneumonia, and laryngotracheobronchitis). As with any vaccine, immediate-type hypersensitivity reactions may occur, including urticaria, anaphylactic reactions, or other immunoglobulin (Ig) E-mediated reactions. MAAEs were collected during the acute phase of either vaccination using the same procedure as for other AEs. SAEs were recorded throughout the study period of the participants' participation. Participants' parents / guardians / legally authorized representatives were asked to immediately notify the study site of any potential SAEs at any time during the study period. Additionally, participants' parents / guardians / legally authorized representatives recorded information about SAEs at the DC / eDC regarding visits from D0 to D56 (cohorts 1 and 3) and visits from D0 to D84 (cohorts 2 and 4). The participants' parents / guardians / legally authorized representatives received a memory aid (MA) to record SAEs from the visit on day 56 until the end of the study (cohorts 1 and 3) and from the visit on day 84 until the end of the study (cohorts 2 and 4). At each visit, the participants' parents / guardians / legally authorized representatives reviewed the completed DC / eDC or MA.

[0146] Reasons for the research design For this Phase I / II trial, data will be analyzed based on serological status. Baseline serological status will be determined retrospectively from serum samples collected at baseline. Participants will be categorized as having experienced RSV or not having been infected with RSV based on the presence or absence of detected serum RSV anti-F IgA antibodies. This biomarker was chosen because it is produced only in response to RSV infection and does not transfer from mother to child across the placenta.

[0147] Post-vaccination immunization assessment on day 56 (cohorts 1, 2, 3, and 4) and day 84 (cohorts 2 and 4): To date, all studies of live attenuated RSV vaccine candidates have used day 56 as the time point for assessing serological antibody responses following a single-dose vaccination. This time point is used for two reasons: 1) because this is a primary response (not a recall response), the serological antibody response may not have reached its maximum level by day 28, and 2) the maximum replication of these highly attenuated vaccine viruses typically does not occur until day 7, which may also delay the induction of an immune response. In subjects receiving two doses of the vaccine, this is no longer a primary response but a recall response, and the serological antibody response is likely to be at its maximum on day 28 after the second vaccination.

[0148] RSV seasonal or post-vaccination RSV monitoring in cohort 4: Based on previous data on RSV seasonality in the United States, Canada, Chile, Honduras, and South Africa, surveillance of RSV-related illness was primarily conducted during the local RSV seasons (November 1 to March 31) and (May 1 to September 30), adjusted for local RSV seasonality. Due to the disruption of global RSV seasonality patterns, it remains unclear when normal seasonal transmission will resume, but there are indications of a rebound in the NH winter of 2021–2022. Due to the unpredictability of the RSV season caused by the impact of non-pharmacological interventions for COVID-19, enrollment and vaccination continued regardless of RSV activity. Study participants enrolled before the regular winter RSV season in the region were visited until the month following the end of the regular season (i.e., April for the NH and October for the SH). Study participants enrolled during the regular RSV season (i.e., November to March for the NH and May to September for the SH) were followed up for at least 5 months after their last vaccination. During RSV season or post-vaccination RSV surveillance, study participants will be monitored for symptomatic respiratory illness requiring medical attention. Note that this surveillance may overlap with the acute and post-acute phases. In such cases, assessments will be required for each relevant phase of the study.

[0149] The dosage of both vaccines is 5.6 log. 10 and 6.2 log 10 PFU assessment: Previous Phase Ia studies conducted by the NIH have shown that 10 6 The RSV ΔNS2 / Δ1313 / I1314L (NIH) vaccine induced a 10% or higher risk of death compared to the previous 10-year PFU dose. 5 PFU doses resulted in better infectivity and neutralizing antibody serum response, while there was no evidence that excessive respiratory illness was associated with receiving higher doses (10). 6 The vaccine was associated with PFU (probiotics, fecal microenzymes, and acetaminophen). However, in these studies, the vaccine was administered intranasally as a nasal drop using a sterile, needle-free 1 mL oral syringe at a volume of 0.5 mL (approximately 0.25 mL per nostril). In this study, the vaccine was administered as a fine nasal mist via an MAD Nasal 130 nasal nebulizer at a total volume of 0.2 mL (approximately 0.1 mL per nostril), requiring reassessment. 5 PFU dosage and 10 6 Safety of both PFU doses when administered via intranasal nebulizer. Use of an intranasal nebulizer can improve overall vaccine delivery and increase replication of both doses. Nebulizer use can also improve the safety of low-dose (10) PFU administration. 5The infectivity of PFU. 10 5 PFU and 10 6 The PFU dose was the minimum dose explored using the MAD Nasal 130 device. The target was at least 5.6 log [amount missing]. 10 PFU and 6.2 log 10 The PFU level is so high, and therefore the dosage is likely to be even higher due to manufacturing requirements / variability.

[0150] Evaluation of two administration methods for vaccine dosage: Several published studies have evaluated the administration of two or three doses of attenuated RSV or type 3 parainfluenza virus (PIV3) candidate vaccines. Generally, the second administration is more restrictive, with a limited serological immune response. Its primary role is to enhance vaccine infectivity in individuals who did not respond to the first administration. Both administrations of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) can increase infectivity at either dose.

[0151] Regardless of baseline RSV serological status, vaccine dosage is selected based on safety, infectivity, viral shedding, and immunogenicity in infants and young children. This includes a non-blinded interim analysis of dose selection for the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine in subjects from cohorts 1, 2, and 3, and in at least 90 subjects enrolled in cohort 4. Interim analyses were conducted when subjects provided safety data up to time point D84 and D84 immunogenicity results were available. Although RSV seronegative infants and young children were likely not previously infected with RSV and exhibited adequate vaccine viral infectivity, shedding, and immunogenicity (based on serum neutralizing antibody titers); vaccine doses were selected based on benefit: risk assessment based on data from the target population of this vaccine (i.e., all infants and young children, regardless of serological status). The selected doses will be used in future studies.

[0152] Placebo group: Each cohort included placebo recipients to determine the background incidence of respiratory and febrile illnesses in infants and young children.

[0153] Clinical hypothesis: Both doses of the attenuated live RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine are expected to be safe and immunogenic for infants and young children. However, administration of either dose may be associated with adverse events that will only become apparent if analyzed in a larger number of subjects than those in the Phase Ia studies enrolled in the Infectious Disease Laboratory (LID) / National Institute of Allergy and Infectious Diseases (NIAID) / NIH (where each study evaluated 10 to 40 vaccine recipients). Furthermore, the rate, magnitude, and duration of the antibody response may vary depending on the dose and the magnitude of the memory (recall) serological response observed following naturally occurring RSV infection. The intranasal nebulizer device used in this study may also affect the safety and immunogenicity of the vaccine. Future dose selection will be based on descriptive comparisons of the safety, infectivity, and immunogenicity of one or two doses of either dose class.

[0154] Security Plan: †All screened subjects provided up to 5 mL of blood sample at enrollment (visit 01) prior to the first administration of the designated investigational product for baseline RSV serum antibody testing.

[0155] *All subjects provided up to 5 mL of blood samples at their visit on D56 (before vaccination 2) and then at their visit on D84 for the purpose of measuring post-vaccination serum antibodies against RSV.

[0156] ‡All subjects provided up to 5 mL of blood samples during one month after the RSV season or at least five months after the last vaccination to measure the post-season RSV antibody titer, thereby determining whether a 4-fold or greater increase in RSV antibody titer had occurred during the RSV season.

[0157] Medical Treatment Procedures Personal medical consultation Visit 01 (Day 0) of queues 1, 2, 3 and 4 The timeline for enrollment completion (visit 01), vaccination, and RSV monitoring for each hemisphere is shown in Table 4. Outpatient Visit 02: Outpatient procedures on day 7 post-vaccination for cohorts 1, 2, 3, and 4 (all subjects) 1) Review the patient's medical history, including past and ongoing medication treatments since the last visit.

[0158] 2) Conduct thorough clinical examinations, including temperature, pulse and respiration, ear, eye, nose, throat (EENT), respiratory, heart and lymphatic systems.

[0159] 3) Obtain nasal swab samples for viral quantification of vaccine-shedded virus. If the subject meets the criteria for seeking medical attention on day 7, the same nasal swab sample can also be used for respiratory pathogen testing.

[0160] 4) Review the responses to requests and proactively provided AEs, AESIs, MAAEs, and SAEs in the completed DC1 / eDC with the subject's parents / guardians / legally authorized representatives. Provide instructions for completing DC2.

[0161] 5) If an SAE occurs, please follow the reporting instructions. Please note that if an SAE occurs, please notify the website.

[0162] 6) Complete the relevant CRF for this visit.

[0163] 7) Remind your parents / guardians / legally acceptable representatives to make the following medical appointment.

[0164] Visit 03: Procedures for patients in cohorts 1 and 3 on day 56+7 after vaccination. 1) Review the patient's medical history, including past and ongoing medication treatments since the last visit. Check for any contraindications.

[0165] 2) Conduct thorough clinical examinations, including body temperature, pulse and respiration, EENT, respiratory system, heart and lymphatic system.

[0166] 3) Obtain blood samples for RSV serum antibodies.

[0167] 4) Collect and review the completed SAEs in DC1 and DC2 / eDC together with the subject's parents / guardians / legally authorized representatives. Provide MA.

[0168] 5) If an SAE occurs, please report it according to the instructions here. Please note that if an SAE occurs, please notify the website.

[0169] 6) Complete the relevant CRF for this visit.

[0170] 7) Remind your parents / guardians / legally authorized representatives to make the following medical appointments.

[0171] Visit 03: Procedure for patients in cohorts 2 and 4 on day 56+7 after vaccination. Obtain the subject's verbal medical history, including past and ongoing drug treatments and immunizations.

[0172] Check for any contraindications to receiving a second vaccination.

[0173] Prior to vaccination, a thorough clinical examination is conducted, including body temperature, pulse and respiration, EENT, respiratory system, heart and lymphatic system.

[0174] Perform COVID-19 POC diagnostic testing.

[0175] Collect reportable concomitant medications / vaccinations.

[0176] Please contact IRT for a dose number.

[0177] Obtain blood samples containing RSV serum antibodies.

[0178] Administer the appropriate investigational product, following the instructions provided for intranasal administration of the investigational vaccine using the MAD Nasal 130 device.

[0179] Observe the subjects for 30 minutes and record any adverse reactions in the original case file.

[0180] Collect and review the responses to requests and proactively raised adverse events (AEs) with the subject’s parents / guardians / legally authorized representatives, including AESI, MAAE, and SAE in completed DC1 and DC2 / eDC.

[0181] 11) Provide or download the Diary Card (DC3) / eDC and thermometer to the parent / guardian / legally authorized representative, and carefully read the instructions for use, reminding them to fill in the pages for Day 0 to Day 7 of the DC3 / eDC. The parent / guardian / legally authorized representative will record the child's temperature and symptoms (requested responses and proactively provided AEs, including AES1, MAAE, and SAE) on the DC3 / eDC.

[0182] 12) Remind parents / guardians / legally authorized representatives to wait for a daily phone call from the research website nurse after this appointment 04 to review what they have entered in DC3 / eDC each day, and to fill out the DC3 entry and bring it to appointment 05 on the specified date and time.

[0183] 13) Remind parents / guardians / legally authorized representatives to notify the website in the event of SAE or any illness. If necessary, website staff will arrange medical treatment within the time specified in the website notification after assessment.

[0184] 14) Complete the relevant CRF for this visit.

[0185] ≥5 months after the RSV season or after the last vaccination appointment for all cohorts (appointment 04 for cohorts 1 and 3, or appointment 06 for cohorts 2 and 4). 1) Obtain blood samples for RSV serum antibodies.

[0186] 2) Review the MA of SAE together with the subject's parents / guardians / legally authorized representatives.

[0187] 3) If an SAE occurs, please report it according to the instructions here. Please note that if an SAE occurs, please notify the website.

[0188] 4) Complete the relevant CRF for this visit and the study termination CRF.

[0189] Visit 04: Procedure for patients in Cohorts 2 and 4 on day 63+7 after vaccination 1 (day 7 after vaccination 2) 1) Review the patient's medical history, including past and ongoing medication treatments since the last visit.

[0190] 2) Conduct thorough clinical examinations, including body temperature, pulse and respiration, EENT, respiratory system, heart and lymphatic system.

[0191] 3) Obtain nasal swab samples to quantify vaccine virus shedding. If the subject meets the criteria for medical attention, the same nasal swab samples can also be tested for respiratory pathogens.

[0192] 4) Review the responses to requests and proactively provided AEs, AESIs, MAAEs, and SAEs in the completed DC3 / eDC with the subject’s parents / guardians / legally authorized representatives.

[0193] 5) If an SAE occurs, please report it according to the instructions here. Please note that if an SAE occurs, please notify the website.

[0194] 6) Complete the relevant CRF for this visit.

[0195] Visit 05: Procedure for patients in cohorts 2 and 4 on day 84+8 after vaccination 1 (day 28+8 after vaccination 2) 1) Review the patient's medical history, including past and ongoing medications and immunizations since the last visit.

[0196] 2) Conduct thorough clinical examinations, including body temperature, pulse and respiration, EENT, respiratory system, heart and lymphatic system.

[0197] 3) Obtain blood samples for RSV serum antibodies.

[0198] 4) Collect and review the responses to requests and proactively provided AEs, AESIs, MAAEs, and SAEs in the completed DC3 / eDC with the subject's parents / guardians / legally authorized representatives. Provide MA.

[0199] 5) If an SAE occurs, please report it according to the instructions here. Please note that if an SAE occurs, please notify the website.

[0200] 6) Complete the relevant CRF for this visit.

[0201] 99 Medical Consultation: Illness Consultation Medical visits can be conducted remotely, or at the investigator's discretion, either at home or in person. The need for medical attention and the type of illness are initially assessed via video call. Video calls are used for a preliminary, remote assessment of the study participant's clinical status and whether the illness can be managed remotely, especially for mild (Level 1) illnesses. During the current COVID-19 pandemic, phased management of medical visits is necessary to limit contact between study participants and research site personnel to only the absolutely necessary level.

[0202] The timeframe for a required medical visit after location notification (if deemed necessary by the investigator) depends on the severity of fever and respiratory symptoms and the stage of the study. If the medical visit occurs on the same day as a routine study visit where a nasal swab sample is to be collected, the same nasal swab sample will be used to test for respiratory pathogens. A second nasal swab should be collected 48 hours later. If the medical visit occurs on the same day as a routine study visit where a nasal swab sample is not collected, a nasal swab sample is required to test for respiratory pathogens. A second nasal swab should be collected 48 hours later.

[0203] In the event of an illness / safety event, study subjects should be managed according to site / local standards of care, including conducting laboratory investigations necessary for the diagnosis and / or management of study subjects. Following illness presentation, healthcare personnel should continue to follow the subject until the condition resolves.

[0204] Medical visits may occur at any time during the study period. Table 5 summarizes the time range for medical visits. LRI, Lower Respiratory Tract Disease; NH, Northern Hemisphere; RSV, Respiratory Syncytial Virus; SAE, Serious Adverse Events; SH, Southern Hemisphere; URI, Upper Respiratory Tract Disease. *In some cases, the acute or post-acute phase may overlap with the RSV monitoring season; in such cases, a more conservative timeframe for seeking medical attention should be considered.

[0205] The medical treatment procedure is as follows: 1) Obtain the subject’s verbal medical history, including past and ongoing drug treatments and immunizations.

[0206] 2) Determine whether the subjects can be managed remotely, or whether home-based medical treatment or online disease consultation is necessary.

[0207] 3) If you are seeking medical treatment at home or in person, please conduct thorough clinical examinations, including temperature, pulse and respiration, EENT, respiratory system, heart and lymphatic system.

[0208] 4) Collect and review, together with the subject’s parents / guardians / legally authorized representatives, the requested and proactively provided AEs, AESI, MAAEs and SAEs in the completed DC / eDC or memory aids.

[0209] 5) If an SAE occurs, please report it as instructed here. If symptoms develop within 28 days of vaccination, remember to notify the location if an SAE occurs.

[0210] 6) Obtain nasal swab samples for RSV virus detection and quantification, as well as real-time reverse transcriptase polymerase chain reaction (rRT-PCR) of respiratory pathogens.

[0211] 7) If necessary, conduct COVID-19 testing based on the opinions of researchers and the CDC COVID testing algorithm (for information on the CDC COVID testing algorithm, see cdc.gov / coronavirus / 2019-ncov / lab / resources / Antigen_Testing_Algorithm_2020-12-14_v03_NO_DRAFT_SPW_508.pdf).

[0212] 8) Complete the relevant CRF for this visit.

[0213] 9) Follow-up visits may be arranged as appropriate, including a visit to the doctor 48 hours later to collect a confirmatory nasal swab.

[0214] 10) In the event of a disease / safety event, the subject should be managed in accordance with the site / local standards of care, including conducting laboratory investigations necessary for the diagnosis and / or management of the study subject.

[0215] Follow up with subjects who respond to requests or experience adverse events leading to the discontinuation of the study / vaccination: Unless the subject or their parent / guardian / legally authorized representative refuses further contact, each subject who experiences an AE (whether severe or mild) during the study must be followed up until the condition resolves, stabilizes, or becomes chronic (even after the subject's participation in the study ends), provided that any of the following is true: 1. Researchers believe that AE is related to the product used.

[0216] 2. AEs (Adverse Events) lead to participants discontinuing the study or stopping vaccination.

[0217] Non-medical consultation telephone contact Acute phase telephone contact to review DCs reaching D29+1 (cohorts 1, 2, 3, and 4) and D84+8 (cohorts 2 and 4). Note: If any acute-phase non-medical contact falls on a weekend or holiday, the call can be made on the next business day. All telephone contacts with the subject's parents / guardians / legally authorized representatives must be made by qualified personnel, such as a doctor or a qualified research nurse.

[0218] 1) Review the response to the request entered into the DC and the information on AE, AESI, MAAE, and SAE proactively provided. After evaluation, website staff will schedule an appointment for the illness within an appropriate timeframe as notified by the location, if necessary.

[0219] 2) Record relevant information concerning the subject's health status, including any changes in medications and immunizations, on the telephone contact form and in the CRF (Clinical Review Form), if necessary. If required, site staff will, after assessment, arrange for the subject to receive medical attention within the timeframe specified in the site notification.

[0220] 3) If an SAE occurs, please report it according to the instructions here. Also, please note that if an SAE occurs, please notify the location.

[0221] 4) Remind parents / guardians / legally authorized representatives to perform the following actions: • Complete the remaining pages of the diary card / electronic diary card and bring them with you at your next appointment.

[0222] • If an SAE occurs, please notify the location.

[0223] Contact by phone on day 42+1 (queues 1, 2, 3 and 4) 1) Record relevant information concerning the subject's health status, including any changes in medications and immunizations, on the telephone contact form and in the CRF (Clinical Review Form), if necessary. If required, site staff will, after assessment, arrange for the subject to receive medical attention within the timeframe specified in the site notification.

[0224] 2) If an SAE occurs, please report it according to the instructions here. Also, please note that if an SAE occurs, please notify the location.

[0225] 3) Remind parents / guardians / legally authorized representatives to perform the following actions: • Complete the remaining pages of the diary card / electronic diary card and bring them with you at your next appointment.

[0226] • If an SAE occurs, please notify the location.

[0227] Telephone contact during RSV season or during RSV surveillance following vaccination. During RSV season (November 1 to March 31 in the Northern Hemisphere and May 1 to September 30 in the Southern Hemisphere) or during RSV monitoring after vaccination (at least 5 months after the last dose of vaccine), contact your parent / guardian / legally authorized representative by phone every 2 weeks.

[0228] 1) Record relevant information concerning the subject's health status on the telephone contact form, including any changes in medications and immunizations.

[0229] 2) For the following types of illnesses requiring medical attention, an appointment should be scheduled according to the guidelines and specified timeframes after contacting the doctor by phone: fever, URI, LRI, or otitis media. If the illness overlaps with an acute or post-acute phase, the timeframe specified for the relevant acute or post-acute phase should be used.

[0230] 3) If a subject has visited any other non-study physician / hospital due to a serious adverse event (SAE) (at any time during the study), a location will be arranged for the subject to obtain a nasal swab sample once the subject is discharged for RSV virus detection and quantification, as well as rRT-PCR against the respiratory pathogen.

[0231] 4) If an SAE occurs, please report it according to the instructions here. Please note that if an SAE occurs, please notify the location.

[0232] Early security data review The sponsor continuously monitors the safety of the research product. To adopt a cautious, phased approach to vaccine administration, an Early Safety Data Review (ESDR) was conducted during the scheduled SMT meeting. • Queue 1 - Post-vaccination D7 ESDR / SMT - Post-vaccination D28 ESDR / SMT - Post-vaccination D56 ESDR / SMT • Queue 2 - Post-vaccination D7 ESDR / SMT - Post-vaccination D28 ESDR / SMT - Post-vaccination D84 ESDR / SMT • Queue 3 - Post-vaccination D7 ESDR / SMT - Post-vaccination D28 ESDR / SMT - Post-vaccination D56 ESDR / SMT • Queue 4 - Post-vaccination D7 ESDR / SMT - Post-vaccination D28 ESDR / SMT - Post-vaccination D84 ESDR / SMT The collected security data was entered into the CRB, and the sponsor compiled each ESDR in a blinded manner. ESDRs were performed by the sponsor during the SMT conference. Group enrollment was not suspended during the SMT review.

[0233] It is understood that all reviews were based on preliminary data that was neither verified nor database-locked. In addition to the security signals specified in the early security analysis during the mid-term of the program definition, the routine and ongoing security signal monitoring process will remain unchanged.

[0234] If a predetermined alarm threshold for a safety event is reached at any other point in the study, enrollment will be suspended regardless of the study time point, in order to convene an ad hoc SMT.

[0235] During safety assessments, Bayesian methods based on posterior distributions can be used to evaluate the differences between each RSV formulation and placebo on the following safety endpoints: • Any grade 3 lower respiratory tract disease, such as wheezing, pneumonia, persistent tachypnea, laryngotracheobronchitis • Level 3 fever The probability that the percentage difference in events between an RSV formulation and a placebo is greater than a pre-specified margin (δ) can be calculated based on a posterior distribution using an uninformed prior β(1,1) and observed binomial data: If this probability is high (e.g., >= 80%), then it may be advisable to abandon the formulation. Therefore, it can help in making decisions regarding security assessments conducted via SMT and / or IDMC (if applicable), and can also be applied at the end of the study.

[0236] If the review of data conducted via SMT does not lead to the identification of potential safety signals, SMT supervision shall be carried out as specified in the SMT bylaws. When safety signals are identified, SMT shall investigate one or more incidents, complete a safety analysis and / or assessment report, and make decisions regarding the safety signals and further actions and / or recommendations, including: • Extend or suspend the trial and report it to the Vaccine Decision Committee (VAC). • Investigate and prepare reports • It is recommended to continue the trial. During the investigation, the team can consult non-clinical safety experts and research team members to identify potential mechanisms underlying the event, and review toxicology data or consult other internal experts as needed.

[0237] If disagreements arise within the team, the core team will seek advice from the VAC (Vaccine Pharmacovigilance Unit) shortly after the SMT meeting, focusing on their respective functional management. Consultation can also be sought with the head of the Vaccine Pharmacovigilance Global Business Unit (PV GBU) or the head of Clinical and Scientific Affairs for the Vaccine GBU, especially if there are issues with the escalation of signals related to the PSB review.

[0238] Once the SMT core team identifies a safety signal that could potentially affect participant safety, study progress (suspension, extended suspension, or termination), or study design (required modifications), the SMT PM notifies the Vaccine PV GBU lead, who verifies the signal and immediately contacts the Vaccine GBU Clinical and Scientific Lead and the PSB Chair to decide whether to convene a PSB meeting.

[0239] Based on the results and recommendations from the PSB, the SMT may implement any of the following operational actions related to the conduct and supervision of the study, including: • Continue experimentation • Extend the suspension period for further evaluation, such as a temporary IDMC. • Modify the experimental design, or • Stop the experiment.

[0240] The interim IDMC (preferably including at least two pediatric respiratory virus vaccine experts) will review the data according to the requirements of the SMT non-blinded study. The interim IDMC will review relevant safety information and available data, including the extent of vaccine virus shedding, as required. If an interim IDMC meeting is required, the interim IDMC will review the available safety data at that point in time to determine whether it is attributable to an etiology, cause, or diagnosis unrelated to the study vaccine, and whether one or more adverse events are associated with vaccine virus shedding at that point in time (even if another is identified). If the interim IDMC concludes that there is no proven causal relationship between the adverse event and the study vaccine, the study can continue. Based on the IDMC's assessment, the sponsoring SMT will determine whether a causal relationship exists between the adverse event and the study vaccine, and then the SMT will decide whether: • Perform internal signal detection procedure (SOP # RDWIN-000390) • Upgrade the verified signal to PSB as needed. • Continue experimentation • Modify the experimental design (using PSB and temporary IDMC inputs) or, • Stop the experiment (using PSB and temporary IDMC input).

[0241] As part of an early security review, the following security parameters were evaluated: • Immediate response • Causes respiratory and systemic reactions • Proactively provided responses, i.e., researcher-reported relevant adverse events (AEs). • SAE, MAAE, and AESI Enrollment was not suspended during the review period. The following checks were performed on the data: 1) Any death, regardless of causality.

[0242] 2) Any SAE related to vaccines.

[0243] 3) Two subjects reported having a grade 3 fever.

[0244] 4) Any adverse event of grade 3 or above, other than fever.

[0245] 5) One subject experienced a lower respiratory tract illness of grade 2 on the DAIDS scale (other than wheezing according to the Brighton Collaboration Severity Scale) during the acute phase (i.e., within 28 days after vaccination).

[0246] 6) Any subject who experiences a grade 3 or higher AESI (other than wheezing according to the Brighton Collaboration Severity Scale) during the acute phase (i.e., within 28 days after vaccination), including lower respiratory tract disease.

[0247] 7)> One subject experienced a Grade 3 active adverse event during the acute phase (i.e., within 28 days after vaccination).

[0248] 8) Any other laboratory values ​​or clinical symptoms, other than fever, that the researchers believe pose a significant safety concern.

[0249] If any of the above criteria are met, it will be determined whether to allow the resumption of enrollment in the study.

[0250] If necessary, case unblinding can be performed, such as as determined by SMT.

[0251] Enrollment and retention of the study group Recruitment process Before the study begins, researchers and / or investigators determine the recruitment strategies to be used at their locations (e.g., advertising, databases, direct mail, or word-of-mouth referrals). They use relevant methods to contact suitable potential parents / guardians / legally authorized representatives and invite them to participate in the study. These locations ensure that any materials used to recruit participants (e.g., information brochures, letters, booklets, posters, other advertisements, etc.) are submitted to the sponsor before being submitted to the IRB for approval.

[0252] In addition, parents / guardians / legally authorized representatives who bring children to the study site for routine medical visits are invited to enroll participants in the study, provided they meet the eligibility criteria. Participants are also recruited from the general population.

[0253] Informed consent procedure Informed consent is a process through which a participant's guardian or appropriate legally acceptable representative voluntarily confirms his or her willingness to participate in a specific research study. Informed consent must be obtained before any research procedure is conducted. This process is documented in writing, signed, and dated by the ICF.

[0254] According to GCP, before the consent form is signed and dated, the subject's guardian / representative must be informed by the appropriate researcher of all aspects of the study relating to the decision to participate, and must have sufficient time and opportunity to ask any questions.

[0255] If the subject's guardian / legally authorized representative is unable to read and sign the ICF, it must be signed and dated by an impartial witness unrelated to the researcher. The witness who signs and dates the consent form testifies that the information in the consent form and any other written information has been accurately explained to and understood by the subject or his / her guardian / representative.

[0256] The ICF actually used by each center may vary, depending on local regulations and IEC / IRB requirements. However, all versions must include the standard information from the sample ICF provided by the sponsor. Any changes to the ICF content must be approved by the sponsor and IEC / IRB before the form is used.

[0257] If new information arises that may relate to the participant's guardian / legally authorized representative's willingness to continue participating in the study, it should be communicated to him / her promptly. Such information is provided through the revised ICF or an appendix to the original ICF.

[0258] Provide two copies of the informed consent form, or a photocopy of the signed consent form. The original should be kept by the researcher, and the copy should be kept by the subject's guardian / legally authorized representative.

[0259] The consent process is documented in the original file.

[0260] Reasons for including participants who were unable to give consent: The basic principles for conducting this study in the pediatric population are as described above.

[0261] Screening criteria Aside from the inclusion and exclusion criteria, there are no other screening criteria. Researchers must review the inclusion and exclusion criteria (visit 01) at the time of registration.

[0262] Inclusion criteria: Individuals must meet all of the following criteria to be eligible to enroll in the study at the time of their initial medical visit: 1. At D0, the age is 6 to 18 months. (6 to 18 months refers to the period from the 6th month after birth to the day before the 19th month after birth.) 2. The informed consent form has been signed and dated by the parent / guardian / or other legally authorized representative (or by an independent witness if required by local regulations).

[0263] 3. Subjects and their parents / guardians / legally authorized representatives are able to attend all scheduled appointments and comply with all trial procedures.

[0264] Exclusion criteria: Individuals who meet any of the following criteria will be excluded from trial enrollment: 1. At the time of study enrollment (or within 6 weeks prior to the first trial vaccination), the candidate was involved in or planned to be involved in another clinical trial of an experimental vaccine, drug, therapeutic device, or treatment procedure during the current study period.

[0265] 2. Received any of the following vaccines prior to enrollment: • You have received any flu vaccine within the previous 7 days, or • Anyone who has received any inactivated vaccine within the previous 14 days, or • Those who received a live attenuated rotavirus vaccine within the previous 14 days, or • Have received any live vaccine (except rotavirus vaccine) within the previous 28 days, or • Have received another investigational vaccine or taken an investigational drug within the previous 28 days.

[0266] 3. Previously received licensed or investigational RSV vaccine or previously received or planned to receive any anti-RSV product (such as ribavirin or RSV immunoglobulin [IG] or RSV monoclonal antibody).

[0267] 4. Received immunoglobulin, blood, or blood-derived products within the past 6 months prior to enrollment.

[0268] 5. Known or suspected congenital or acquired immunodeficiency; or having received immunosuppressive therapy, such as chemotherapy or radiotherapy for cancer, within the past 6 months; or having long-term systemic corticosteroid therapy (using prednisone or its equivalent for more than 2 consecutive weeks within the past 3 months).

[0269] 6. Possible or confirmed cases of COVID-19.

[0270] 7. Known systemic hypersensitivity to any component of the vaccine, or a history of life-threatening reactions to the vaccine used in the trial or to any vaccine containing the same substance.

[0271] 8. Any chronic disease.

[0272] • Chronic diseases may include, but are not limited to, heart disorders, lung diseases (including reactive airway diseases, any history of wheezing treated with bronchodilators or a medical diagnosis), kidney disorders, autoimmune disorders, diabetes, psychomotor disorders, and known congenital or hereditary diseases. 9. Any history of wheezing diagnosed by a medical institution.

[0273] 10. Any acute fever, respiratory, or gastrointestinal illness within the past 24 hours that, in the investigator's opinion, is severe enough to interfere with successful vaccination on the day of vaccination. Potential subjects should not be included in the study until the symptoms subside or the fever subsides.

[0274] 11. Received any of the following medications within 3 days prior to study enrollment: • Systemic antibacterial, antiviral, antifungal, antiparasitic, or antituberculosis drugs, whether for treatment or prevention, or • Intranasal medication, or • Other prescription medications besides licensed companion medications (prescription or nonprescription) include nutritional supplements, medications for gastroesophageal reflux, eye drops, and topical medications, including (but not limited to) topical steroids, topical antibiotics, and topical antifungals.

[0275] 12. Received salicylates (aspirin) or products containing salicylates within 28 days prior to enrollment.

[0276] 13. Deprivation of liberty in emergency situations or during involuntary hospitalization.

[0277] 14. The biological or adopted children of researchers or employees who are identified as being directly involved in the proposed study.

[0278] 15. Any previous allergic reactions.

[0279] 16. Any prior vaccine-related grade 3 or higher adverse reaction. Note: If a grade cannot be determined, determine if the reaction is serious or life-threatening; if so, it is exclusionary.

[0280] 17. Families that include or will include members of families with infants under 6 months of age between the enrollment date (or within 6 weeks prior to the first trial vaccination) and day 28.

[0281] 18. A member of another child / family of another child who was enrolled or is planned to enroll in the study in the same year, and whose enrollment date will not be the same as that of other participants living in the same household (i.e., all eligible children from the same household must be enrolled on the same date).

[0282] 19. Members of families containing immunocompromised individuals, including but not limited to: • People living with HIV • People who received chemotherapy within 12 months prior to enrollment • People receiving immunosuppressants • People who have received solid organ or bone marrow transplants.

[0283] 20. Attending a daycare facility and sharing a daycare room with an infant under 6 months of age, and the parents / guardians / legally authorized representative are unable or unwilling to suspend daycare for 28 days after vaccination.

[0284] 21. Following the planned vaccination, administer the following as scheduled: • Get any flu vaccine within 7 days afterward, or • Within the following 14 days, administer an inactivated vaccine or a live attenuated rotavirus vaccine, or • Within the following 28 days, receive any live vaccine other than rotavirus, or • Receive another investigational vaccine or take an investigational drug within the next 56 days.

[0285] 22. Born before 34 weeks of gestation.

[0286] 23. Born before 37 weeks of gestation and under 1 year old at enrollment.

[0287] 24. Currently suspected or confirmed to have developmental disorders, developmental delays or other developmental problems.

[0288] 25. Has previously received any supplemental oxygen therapy in a home or hospital setting, except in cases of temporary supplemental oxygen therapy due to transient tachypnea in newborns.

[0289] Medical history Prior to enrollment, subjects' pre-existing conditions and illnesses are assessed, including past and ongoing ones. Any such conditions are documented in the source document. Significant (clinically relevant) medical history (reported as a diagnosis) includes conditions / illnesses that physicians have described or have described during their visits to or visits to subjects, or conditions / illnesses collected in the CRB that may recur or cause SAEs or require repeat outpatient care during the study. The CRB's Significant Medical History section contains a core list of bodily systems and disorders, which can be used to facilitate comprehensive reporting as well as space for reporting specific conditions and illnesses.

[0290] For each condition, the data collected is limited to: • Diagnosis (this is better than reporting signs and symptoms) • Was the disease present at the time of enrollment? • It is strongly recommended that you do not substitute reporting signs and symptoms for diagnosis.

[0291] Dates, medications, and body systems will not be recorded, and the information collected will not be coded. The purpose of this limited data is to aid in the later interpretation of safety data collected during the study.

[0292] Contraindications for subsequent vaccinations Temporary contraindications If a subject experiences one of the following symptoms on day 0 (cohort 1, cohort 2, cohort 3, and cohort 4) or day 56 (cohort 2 and cohort 4), the investigator will postpone further vaccination until the symptoms subside. The postponement must still fall within the timeframe intended for vaccination, i.e., within 5 days after randomization on day 0 (cohort 1, cohort 2, cohort 3, and cohort 4) and within 7 days after day 56 (cohort 2 and cohort 4).

[0293] According to the investigator’s judgment, any acute febrile illness (rectal temperature ≥38.0°C [≥100.4°F]), acute otitis media, upper and lower respiratory tract signs or symptoms (including but not limited to nasal discharge, cough and pharyngitis) or nasal obstruction within the past 24 hours that is severe enough to interfere with the successful absorption of the study product.

[0294] Administered before or after any study vaccination, with any of the following: • You have received any flu vaccine within the previous 7 days, or • Anyone who has received any inactivated or live attenuated rotavirus vaccine within the past 14 days, or • Have received any live vaccine (except rotavirus vaccine) within the previous 28 days, or • Have received another investigational vaccine or taken an investigational drug within the previous 28 days.

[0295] All eligible subjects from the same family enrolled on the same date must receive the study product on the same date, and therefore, if one child experiences the disease described above, product administration should be postponed for both children.

[0296] Clear contraindications The researchers will suspend vaccination if the participant develops any of the following conditions: • An allergic reaction to a previous dose of the vaccine or any other significant allergic reaction.

[0297] • Any AE greater than grade 2, any level of LRI, or investigator-assessed SAE related to a previous vaccine dose.

[0298] • Diagnosis of COVID-19 If applicable, subjects with clear contraindications should be followed up for study-defined safety and immunogenicity assessments.

[0299] In the case of local or national immunization programs using pandemic vaccines (e.g., influenza), subjects who receive a pandemic vaccine at any time during the study period should withdraw from the study.

[0300] Exit conditions Parents / guardians / legally authorized representatives are informed of their right to withdraw their child from the study at any time. Any participants who have accepted the study product are encouraged to continue with study follow-ups for the duration of the study, even if sample collection is refused.

[0301] Participants can withdraw from the study: • Upon oral or written request from the parent / guardian / legally authorized representative (i.e., withdrawal of consent / withdrawal).

[0302] • Due to safety considerations regarding serious non-compliance with the protocol without the permission of a parent / guardian / legally authorized representative (i.e., non-compliance / withdrawal), this is determined at the sole discretion of the researcher or sponsor.

[0303] The reasons for withdrawal or exit should be clearly documented in the source file and CRB.

[0304] Researchers must determine whether voluntary withdrawal is due to safety concerns (in which case the reason for withdrawal is noted as "adverse event") or for other reasons.

[0305] Those who withdraw will be replaced.

[0306] For any participant who withdraws or terminates the study before the planned visit is completed, researchers document the reasons for withdrawal or termination in detail and make every effort to complete the following final assessment: Obtain the subject's verbal medical history, including past and ongoing drug treatments and immunizations.

[0307] If possible, obtain a blood sample containing RSV serum antibodies.

[0308] Nasal swab samples were obtained to quantify vaccine virus shedding (if in the acute phase), other respiratory pathogens, including investigator-determined COVID-19 POC testing.

[0309] Complete the relevant parts of the CRF.

[0310] Loss to follow-up procedures In cases where subjects are unable to return for follow-up examinations, documented and reasonable efforts (i.e., documented telephone calls and registered mail) are made to locate or recall them, or at least to determine their health status while fully respecting their rights. These efforts are documented in the source file.

[0311] Classification of subjects who discontinued the study For any participant who discontinues the study before completion, the most important reason for early termination will be examined in the CRB. The reasons are listed below in descending order of importance: Discontinued follow-up All scheduled safety follow-ups were completed at this site, and any participants who terminated the study early due to adverse events (AEs) or protocol deviations were contacted.

[0312] For subjects whose reasons for early termination could not be traced, or if a subject withdrew informed consent and explicitly stated that they did not wish to be contacted again and this was documented in the source file, the location did not attempt to obtain further safety information.

[0313] If a participant’s status at the end of the study is “Participant or parent / guardian / legally authorized representative withdraws,” the location will attempt to contact them for a post-RSV season appointment unless they explicitly state that they do not wish to be contacted again and this is documented in the source file.

[0314] Follow-up of participants in the study who had COVID-19 If a participant develops COVID-19 during the study, they will be followed up for as long as possible in accordance with the guidelines of the national / regional / local health authorities. All efforts are made to monitor the safety of study participants and collect key samples, while adhering to the guidelines of the national / regional / local health authorities.

[0315] Security Emergency Phone If, at the researcher's discretion, a participant experiences a medical emergency, the researcher may contact the sponsor's RMO for advice on how to resolve any research-related medical questions or issues. If the RMO is unavailable, the researcher may contact the call center (available 24 / 7), which will, as needed, transfer all security emergency calls to the appropriate primary or backup sponsor contact.

[0316] This process does not replace the need to report SAEs. Researchers are still required to follow the protocol-defined procedures to report SAEs to the Global Pharmacovigilance (GPV) department.

[0317] In the event of an emergency unblinding, researchers must follow the unblinding procedure.

[0318] Research and revision of the plan Any modifications to this research plan and protocol must be discussed with and approved by the sponsor. If the necessity of a revision is agreed upon, it shall be submitted in writing by the sponsor, and the revised version of the agreement shall supersede the earlier version. All substantive revisions (e.g., those affecting the conduct of the study or the safety of the participants) require IEC / IRB approval and must also be forwarded to the regulatory body.

[0319] Administrative revisions to the protocol refer to modifications to some administrative, logistical, or other aspects of the study that do not affect its scientific quality or the safety of the participants. Only notification to the IEC / IRB is required; formal approval is not necessary.

[0320] Researchers are responsible for ensuring that, during the period for which IEC / IRB approval has been given, no changes to the approved study are initiated without IEC / IRB review and approval, unless it is to eliminate any apparent direct harm to the subjects.

[0321] Research interrupted If new data about the research product generated by this study or any other study becomes available, the study may be suspended; or for administrative reasons; or as advised by the sponsor, researcher, IEC / IRB or the institute’s national regulatory body.

[0322] If the study is terminated or suspended early, the sponsor shall immediately notify the investigator, IEC / IRB, regulatory body, and any one or more contract research organizations used in the study of the reason for the termination or suspension, as specified in applicable regulatory requirements. The investigator shall immediately notify the parents / guardians / legally authorized representatives of the subjects and shall ensure appropriate treatment and / or follow-up for the subjects.

[0323] Products applied Research product identity The characteristics of the research products are described in sections 1 through 4 below.

[0324] Research Product 1's Identity Respiratory syncytial virus (RSV) RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine 5.6 log 10 PFU / 0.2 mL, viral suspension.

[0325] composition Each 0.2 mL dose of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine contains the following components: Attenuated live RSV 5.6 log 10 PFU, which is an attenuated live RSV with (i) a 523-nucleotide deletion in the NS2 gene, (ii) an amino acid deletion in the L protein (Δ1313; deletion of S1313), and (iii) a genetically stable mutation in the L gene (I1314L), is delivered as a droplet mist (size range 10 µm to 120 µm) using an intranasal nebulizer device at approximately 0.1 mL / nostril.

[0326] Preparation and application In the Phase I / II clinical trial, a commercially available intranasal nebulizer (MAD) device was used to administer the experimental RSVΔNS2 / Δ1313 / I1314L (Sanofi) vaccine. The selected device, the MAD130 system, was equipped with a nozzle (MAD 300 nebulizer), a 1 ml plastic syringe, and a plastic vial access cannula. For the Phase I / II clinical study, the sponsor-manufactured experimental RSVΔNS2 / Δ1313 / I1314L (Sanofi) vaccine was filled and stored in vials. At the clinical trial site, the experimental RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine was drawn from the vial into the syringe at the specified volume, the device / nozzle was attached to the syringe head and filled, and the dose was administered intranasally using the MAD130 device, with approximately half the dose given in each nostril.

[0327] Prior to administration, whenever possible, all study products must be visually inspected for cracks, broken seals, incorrect labeling, foreign particulate matter, and / or discoloration, regardless of the solution and container. If any of these conditions are present, the vaccine should not be administered. An alternative dose was used, and the incident was reported to the sponsor.

[0328] Subjects must be observed for 30 minutes after each vaccination to ensure their safety, and any reactions during this period are recorded in the CRB. The website is stocked with appropriate medical equipment and emergency medications, including adrenaline (1:1000), in case of allergic reactions, vasovagal reactions, or other immediate-type anaphylactic reactions.

[0329] Dosage selection and timing Subjects in cohort 1 received a single dose of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 5.6 log on day 0. 10 PFU vaccine.

[0330] Subjects in cohorts 2 and 4 received two administrations: one administration on day 0 of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 5.6 log 10 The PFU vaccine was administered again on day 56.

[0331] Vaccination device A nasal mucosal nebulizer (MAD) manufactured by Teleflex, licensed and commercially available in the United States, was used as the vaccine device. The selected device, the MAD130 system, is equipped with a nozzle (MAD 300 nebulizer), a 1 ml plastic syringe, and a plastic vial access cannula. For Phase I / II clinical trials, the sponsored experimental RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine or control product was filled and stored in vials. At the clinical trial website, the experimental RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine or control product was drawn from the vial into the syringe at the specified volume, the device / nozzle was attached to the syringe head and filled, and the dose was administered intranasally using the MAD130 device, with approximately half the dose given in each nostril.

[0332] The mechanism of action is as follows: When manual pressure is applied to the syringe, the plunger propels the liquid product through the spray nozzle and atomizes the drug product into a spray form. The conical stopper is engineered to guide the spray plume more consistently toward the top of the nasal passage, through the nasal flap, and into the nasal cavity.

[0333] Teleflex MAD Nasal TM Manufacturing information for intranasal mucosal nebulizer devices: Made of radiation-stable medical-grade polycarbonate material, it meets the requirements of USP Class VI and ISO 10993.

[0334] Manufactured in an ISO Class 7 cleanroom environment.

[0335] The manufacturing process complies with FDA 21 CFR Part 820, ISO 13485 and EU MDD.

[0336] It is pyrogen-free and latex-free.

[0337] Teleflex MAD Nasal TM The intranasal mucosal nebulizer conforms to ISO-594 standards and can be used with any ISO-594 compliant Luer lock syringe.

[0338] Manufacturer Specifications: The MAD130 intranasal mucosal nebulizer has the following manufacturer specifications as listed in the product manual: • Typical droplet size: 30-100 μm • System dead zone: 0.15 mL • Tip diameter: 4.3 mm Research Product 2's Identity Respiratory syncytial virus (RSV) RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine 6.2 log 10 PFU / 0.2 mL, viral suspension.

[0339] composition Each 0.2 mL dose of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine contains the following components: Attenuated live RSV 6.2 log 10 PFU, which is an attenuated live RSV with (i) a 523-nucleotide deletion in the NS2 gene, (ii) an amino acid deletion in the L protein (Δ1313; deletion of S1313), and (iii) a genetically stable mutation in the L gene (I1314L), is to be delivered as a fine mist (10 µm to 120 µm in size) using an intranasal nebulizer device at a dose of approximately 0.1 mL / nostril.

[0340] Preparation and application The preparation and administration procedures for the control product are the same as those for the study product.

[0341] Dosage selection and timing Subjects in cohort 3 received a single dose of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 6.2 log on day 0. 10 PFU vaccine.

[0342] Subjects in cohort 4 received two administrations: one on day 0 of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 6.2 log 10 The PFU vaccine was administered again on day 56.

[0343] Vaccination device This article describes a vaccination device.

[0344] Check the product's identity placebo composition The same formulation buffer as the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine is delivered at approximately 0.1 mL per nasal cavity.

[0345] Preparation and application The preparation and administration procedures for the control product are the same as those described for the study product in this paper.

[0346] Dosage selection and timing Subjects in cohort 1 and cohort 3 were given a placebo once on day 0.

[0347] Subjects in cohorts 2 and 4 received two administrations: a placebo was administered once on day 0 and again on day 56.

[0348] Vaccination device This article describes a vaccination device.

[0349] Product Logistics Labels and Packaging Both the investigational product and placebo in individual dose vials are provided with investigational labeling and packaging in accordance with national regulations. Each individual dose of investigational product or placebo is identified by a unique number on the main label and outer carton label. The carton label also includes a removable tag for location affixing to the source document.

[0350] The study product and placebo product were blinded at the carton level.

[0351] Product transportation, storage and liability Product transportation The clinical study manager or designated personnel will contact the investigator or designated personnel to determine the date and time of product delivery.

[0352] Each batch of vaccine shipments is equipped with temperature monitoring devices to verify the maintenance of the cold chain during transportation. Upon delivery to the location, the personnel responsible for receiving the products operate according to instructions, including checking the maintenance of the cold chain during transportation (i.e., verifying the temperature recorder). If there are any indications that the cold chain has been breached, the personnel should immediately isolate the product, notify the sponsor representative, and request authorization from the sponsor to use the product.

[0353] Product Storage The researcher may personally be responsible for product management or designate a staff member to assume this responsibility.

[0354] At the site, the product must be stored in a secure location with restricted access. The vaccine must be stored in a freezer at -60°C (-76°F) and protected from light. Temperature must be monitored and recorded throughout the entire duration of the vaccine's stay at the research site. In the event of an unexpected disruption to the cold chain, the vaccine must not be administered and must be isolated; the researcher or authorized representative should contact the sponsor's representative for further instructions.

[0355] Product Responsibility The personnel responsible for site product management maintain records of product delivery to the study site, site product inventory, one or more doses administered to each participant, and the disposal or return of unused doses to the sponsor. Because vaccines and placebos differ in appearance, one or more unblinded coordinators verify product liability and distribute the vaccine.

[0356] The necessary information for the product label has been entered into the source file and CRB. Where applicable, the information has also been entered into the subject's vaccination card.

[0357] Sponsor monitoring personnel verify product liability records at the study site based on dosage records in the CRB and communications from the IRT (if applicable).

[0358] In the event of any anticipated or potential product shortage during the study, the investigator or an authorized designated person shall promptly notify the sponsor representative and arrange for the delivery of additional doses.

[0359] Alternative dose If a dose replacement is required (e.g., due to a broken syringe or particulate matter observed in the syringe), the site personnel must contact the IRT to accept the new dose dispensing.

[0360] Disposal of unused products Unused or discarded products were returned to the sponsor. Product liability was verified throughout the study.

[0361] Product Recall If the sponsor decides to initiate the search process, one or more researchers will be informed of what needs to be done.

[0362] Blinding and decryption procedures This study was conducted in an observer-blinded manner. • The investigators and researchers conducting the safety assessment, as well as the subjects, were unaware of which vaccine was being administered.

[0363] • Only researchers who prepare and administer the vaccine, and who are not involved in safety assessments, know which vaccine was administered.

[0364] Parents / guardians / legally authorized representatives, researchers and research staff collecting safety data, and laboratory personnel analyzing blood samples are unaware of which product was administered. The vaccination specialist is responsible for preparing and administering the product and has no authority to collect any safety data. Furthermore, the vaccination specialist or authorized designee ensures that randomization documents are stored in a secure location accessible only to them.

[0365] Unblinding may only be performed in the event of an adverse event (AE) if the identification of the received vaccine could affect the subject's treatment. Unblinding should be limited to one or more subjects who experienced an AE.

[0366] Researchers or their representatives can unblind the subject through the IRT system. Once the site has handled the emergency and the subject has been unblinded, the researcher or representative must notify the sponsor, the RMO. All attempts to contact the sponsor prior to unblinding should be documented in the source file, and a CRF for unblinding should be completed.

[0367] The following are also methods for requesting to clear one's eyes: • In the event of an SAE, the GPV department will report to the health authority through its internal system as described in ICH E2A. In this case, only the one or more subjects in question will be unblinded. Information resulting from the code-breaking (i.e., the subject's vaccine or group assignment) will not be communicated to the investigator or the team directly involved in the study, except to the GPV representative.

[0368] • If necessary, by a temporary IDMC to facilitate security assessment.

[0369] The IEC / IRB must be notified of any unblinding. All documents related to the incident must be kept in the site's research records and in the sponsor's files. Any intentional or unintentional unblinding must be reported, documented, and explained, and the name of the person who requested the unblinding must be provided to the sponsor.

[0370] A non-blinded interim analysis is planned for dose selection of the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine in at least 90 subjects from cohorts 1, 2, and 3, as well as in enrolled cohort 4. The interim analysis will be conducted when subjects provide safety data up to time point D84 and D84 immunization results are obtained. This non-blinded interim analysis requires unblinding data; specific procedures have been implemented to maintain blinding at both the subject and investigator levels.

[0371] Tests conducted in the sponsor's and contract laboratories are blinded for the allocation of the study treatment group. One or more codes linking information about the sample vials to the study treatment group assignment are retained by the clinical department and are not accessible to the sponsor's or contract laboratory testing personnel.

[0372] Randomization and Assignment Procedures IRT was performed at the time of consultation to assign subject numbers and to assign vaccine groups with dose numbers to the subjects.

[0373] At the time of consultation 01, subjects who met the inclusion / exclusion criteria and whose parents / guardians / legally authorized representatives had signed the ICF were randomly assigned to one of the vaccine groups according to the cohort: • Queues 1 and 2: RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 5.6 log 10 PFU (low-dose RSV) or placebo in a 1:1 ratio • Queue 3: RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 6.2 log 10PFU (high dose of RSV) or placebo, in a 1:1 ratio • Queue 4: RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 6.2 log 10 PFU (high-dose RSV) or RSV ΔNS2 / Δ1313 / I1314L (Sanofi) 6.2 log10 PFU (low-dose RSV) or placebo, in a ratio of 1:1:1 At the first visit, patients were randomized by cohort and age subgroup (<12 months / ≥12 months).

[0374] Site staff connect to the IRT, enter their identity and security information, and confirm the minimum amount of data according to IRT prompts. The IRT then provides dose number assignments for site staff to confirm. If a subject is ineligible to participate in the study, this information is only recorded in the subject recruitment log.

[0375] The subject number assigned by the IRT consists of a 12-digit string (3-digit country / region identifier, 4-digit research center identifier, and 5-digit subject identifier). For example, subject 840000100005 is the fifth subject registered at Center 1 in the United States (840 is the US country / region code).

[0376] Treatment adherence The following measures ensure that the administered vaccine doses conform to the planned doses, and any discrepancies are documented so that they can be taken into account in data analysis: • All vaccinations were administered by qualified researchers.

[0377] • The personnel responsible for product management at the study site maintain records of responsibility for product delivery to the study site, site product inventory, administration of one or more doses to each subject, and disposal of unused or wasted doses.

[0378] Accompanying medications and other therapies At the time of enrollment, ongoing medications and other therapies (e.g., blood products) and new medications prescribed for new medical conditions / AEs during study participation should be documented in the source file.

[0379] Records of continuing companion medications in the CRB are limited to one or more medications of a specific class that have been in use since the date of the first vaccination. This may include medications of concern that were started before the day of vaccination.

[0380] From the date of each vaccination to the end of the requested and voluntary follow-up period, the CRB collects the reportable medications.

[0381] Reportable drugs include those that affect or may affect the consistency of safety information collected after any vaccination and / or vaccination-induced immune response. Three standard categories of reportable drugs are defined: • Drugs that affect or may affect safety assessments (e.g., antipyretics, analgesics, nonsteroidal anti-inflammatory drugs [NSAIDs], steroids / corticosteroids).

[0382] • Drugs that affect or may affect the immune response (e.g., other vaccines, blood products, antibiotics that may interfere with bioassays used by the GCI department, steroids / corticosteroids, immunosuppressants, immunomodulators with immunosuppressive properties, antiproliferative drugs such as DNA synthesis inhibitors).

[0383] • Medications that affect or may affect safety and immune response (e.g., steroids / corticosteroids) Information on each reported drug in the CRB is limited to: • Product Name • Prescription source: Prevention yes / no. One or more prescribed medications used to prevent AEs are recorded in the "Measures Taken" section of the AE collection form.

[0384] • Start and end dates • Reasons for treatment Dosage and route of administration, homeopathic medications, topical and inhaled steroids, and topical, ophthalmic, and otological treatments were not documented. No topical analgesics were applied to the vaccination site; however, if they were unintentionally applied to the vaccination site, they would be documented as Class 1 medications in such specific circumstances.

[0385] Medications administered for an AE are only documented in the "Actions Taken" section of the AE CRF. No details are documented in the accompanying medication CRF unless one or more of the medications administered fall into one of the pre-listed categories.

[0386] The drug is coded.

[0387] Contraindicated concomitant drugs The following are prohibited: • Prophylactic antipyretics, decongestants, or antihistamines during the acute phase (28 days after administration of the study product) — Note that these medications are permitted for the treatment of symptoms.

[0388] • Use of investigational drugs or investigational vaccines other than the investigational product within 56 days of receiving the investigational product.

[0389] Preventive concomitant medications Because they can potentially confound immunogenicity results, the following treatments should be avoided after administration of investigational products unless clinically indicated: • Systemic corticosteroid therapy for more than 14 days, at a dose equivalent to prednisone >2 mg / kg or 20 mg daily or other immunomodulatory drugs.

[0390] • Immunoglobulins and / or any blood products.

[0391] Unless there is an outbreak of an epidemic, the following should be avoided after the application of research products: • Licensed inactivated or live attenuated rotavirus vaccines administered within 14 days of receiving the investigational product.

[0392] • Licensed live virus vaccines (excluding rotavirus vaccines) administered within 28 days of receiving the investigational product. Sample Management Blood samples for assessing antibody response were collected from subjects in Cohort 1 and Cohort 3 who received a single dose at visits 01, 03, and 04, and from subjects in Cohort 2 and Cohort 4 who received two doses at visits 01, 03, 05, and 06. For detailed information on the sampling schedule, please refer to the study procedure table in this document.

[0393] Nasal swab samples for RSV confirmation were collected from subjects in Cohort 1 and Cohort 3 who received a single dose at visit 02 (day 7), and from subjects in Cohort 2 and Cohort 4 who received two doses at visit 02 (day 7) and visit 04 (day 63). Nasal swab samples were collected from subjects during illness visits for the detection of RSV and respiratory pathogens.

[0394] All sample collections conducted in the United States followed the Centers for Disease Control and Prevention (CDC) interim infection prevention and control recommendations for patients with suspected or confirmed COVID-19 in healthcare settings. Sample collections conducted outside the United States complied with local regulatory guidelines for COVID-19.

[0395] For detailed information on the sampling schedule, please refer to the research procedure table in this article.

[0396] Sample collection During the aforementioned medical visits, up to 5 mL of blood was collected in test tubes provided or recommended by the sponsor.

[0397] During the aforementioned visits, nasal swab samples were collected and transferred to test tubes provided or recommended by the sponsor. Staff collecting the samples implemented appropriate infection prevention and control measures.

[0398] Immediately before drawing blood or a nasal swab, the staff performing the procedure verify the subject's identity and the assigned subject number and sampling stage on a pre-printed label, which is then affixed to the test tube.

[0399] Sample preparation serum samples An overview of the process is provided here.

[0400] After blood is drawn, the test tubes are left to stand upright without shaking for a minimum of 1 hour and a maximum of 24 hours to allow the blood to clot. Samples are kept at room temperature for up to 2 hours; after 2 hours, they must be refrigerated at +2°C to +8°C (+35.6°F to +46.4°F) after the room temperature clotting period and must be centrifuged within a maximum of 24 hours.

[0401] The samples were then centrifuged, and the serum was transferred to an appropriate number of aliquots. These aliquots were pre-labeled with adhesive tags identifying the study code, subject number, and sampling stage or visit number.

[0402] The subject's identification number, sampling date, number of aliquots obtained, preparation date and time, and the subject's consent to future use of his / her sample should be noted on the sample identification list and recorded in the source file. This list provides space for commenting on sample quality.

[0403] nasal swab sample This article provides an overview of the process.

[0404] The subject's identification number and any other required information, the sampling date, and the preparation date and time were clearly recorded.

[0405] Sample storage and transportation serum samples During storage, serum tubes should be kept in a freezer at a temperature set and maintained at -20°C (-4°F) or below. Temperature should be monitored and recorded appropriately throughout the study. The clinical logistics coordinator should be notified if the temperature rises above -10°C (14°F) at any time.

[0406] Goods were only delivered to the laboratory after appropriate monitoring and notification to the clinical logistics coordinator. Serum was transported frozen, using dry ice to maintain it in the frozen state within the carrier-supplied packaging containers. Temperature was monitored again. Shipping must comply with UN Class 6.2 specifications and IATA 602 packaging instructions.

[0407] The samples were transported to the Samples, Reagents and Animal Services division within the sponsor's global R&D operations.

[0408] nasal swab sample Nasal swab samples are transported frozen, using dry ice to keep them frozen in the packaging containers provided by the carrier.

[0409] Stored biological samples will be used for future research. Following the completion of the study, any unused portions of the serum and nasal swab samples will be securely stored at the sponsor's facility for at least 25 years. These samples will be retained for long-term storage to support the resolution of regulatory questions related to product licensing and potential revalidation of study results. Furthermore, these samples will be used in the development of detection methods for RSV or other respiratory pathogens.

[0410] Other biological samples collected to determine participant eligibility or to monitor their health will be used immediately. If they are not fully used, they will be destroyed no later than the end of the study or after the time required by local law.

[0411] In addition, parents / guardians / legally authorized representatives are required to indicate in the ICF whether they permit the future use of any unused stored serum samples for additional testing. If they refuse permission, these samples will not be used for any testing other than those directly related to this study. If they consent to such use, they will not receive compensation for granting permission. Anonymity of the samples is ensured. At present, the purpose of any potential future research is unknown and may be unrelated to this particular study. It could be to improve understanding of vaccines or infectious diseases, or to improve existing tests or develop new tests to evaluate vaccines. Human genetic testing will never be performed on these samples without the specific individual's informed consent.

[0412] Clinical supplies Sponsors provide the research website with protocols, ICF, CRB, SAE reports, diary cards, memory aids, and other research documents, as well as the following research materials: all research vaccines, including vaccine administration devices, blood collection tubes, cryovials, cryovial storage boxes, cryovial labels, temperature recorders, transport containers, and digital thermometers.

[0413] The method for performing Electronic Data Acquisition (EDC) is defined by the sponsor. If the sponsor provides a computer, it is retrieved at the end of the study.

[0414] The researchers provided all vaccination supplies, blood collection equipment, and centrifugation equipment, including biohazard and / or safety supplies. Biohazard and safety supplies included needles and syringes, examination gloves, lab coats, sharps disposal containers, and absorbent tablecloths. The location ensured that all biohazard waste was autoclaved and disposed of according to local practices. The researchers also provided appropriate space in a temperature-monitored refrigerator for storing products and for blood samples, and appropriate space in a temperature-monitored refrigerator for aliquoting serum samples.

[0415] If additional supplies are needed, the researchers will contact the sponsors and specify the required quantity.

[0416] Endpoints and evaluation methods Primary endpoints and evaluation methods Security Security Definition The following definitions are taken from the ICH E2A Clinical Safety Data Management Guidelines: Definitions and Standards for Rapid Reporting.

[0417] Adverse events (AEs): An AE is any unfortunate medical event that occurs in a patient or clinical trial subject who has been given a pharmaceutical product and is not necessarily causally related to that treatment. Therefore, an AE can be any adverse or unexpected sign (including, for example, an abnormal laboratory finding), symptom, or illness that is temporarily associated with the use of the pharmaceutical product, whether or not it is considered to be related to the pharmaceutical product.

[0418] Therefore, AE could be: • New diseases • Pre-existing symptoms worsen • The effectiveness of vaccination, including comparison doses • The above combinations All adverse events (AEs) include both serious and non-serious AEs.

[0419] Surgical procedures are not adverse events (AEs); they are measures taken to treat a medical condition. An AE is a condition that leads to the taking of such measures (if it occurs during the research period).

[0420] Pre-existing medical conditions should not be reported as adverse events (AEs). However, if a pre-existing medical condition worsens in frequency or intensity after the study intervention, or if it changes in its clinical significance according to the investigator, such a change should be reported as an AE (exacerbation). The same applies to recurrent episodes of pre-existing conditions (such as asthma) if the frequency or intensity increases after vaccination.

[0421] Serious adverse events (SAE): “Serious” and “major” are not synonyms. The term “major” is typically used to describe the intensity of a particular event, equivalent to level 3. This differs from “severity,” which is based on criteria for subject / event outcomes or actions typically associated with an event that poses a threat to the life or function of the subject. “Severity,” not “major,” is used as a guideline for defining regulatory reporting obligations.

[0422] SAE is any unfortunate medical event that results in the following outcomes at any dose: • Resulted in death • Life-threatening • Requires hospitalization or extension of existing hospital stay • Leading to persistent or significant disability / incapacity • It is a congenital malformation / birth defect • It is an Important Medical Event (IME) The term "life-threatening" refers to an event in which the subject faces a risk of death when the event occurs; it does not refer to an event that, hypothetically, could lead to death if it were more severe.

[0423] All medical events leading to hospitalization were recorded and reported as SAEs, except for the following: planned hospitalizations prior to inclusion in the study or outpatient treatments that did not require hospitalization.

[0424] "Persistent or significant disability or incapacity" refers to a severe impairment of a person's ability to function normally in daily life.

[0425] Medical and scientific judgment should be applied when determining whether expedited reporting is appropriate in other circumstances (such as IMEs that may not be immediately life-threatening or result in death or hospitalization, but may endanger the health of the subject or may require intervention to prevent one of the other outcomes listed in the definition above). These IMEs should generally also be considered serious. Examples of such events include allergic bronchospasm requiring intensive treatment in the emergency room or at home, blood cachexia or seizures that do not lead to hospitalization, or the onset of drug dependence or abuse, new-onset diabetes, or autoimmune diseases.

[0426] Adverse reactions: All harmful and unexpected responses to any dose of a drug should be considered adverse reactions (ARs).

[0427] (The phrase "response to a drug" means that there is at least a reasonable probability of a causal relationship between the drug and the adverse event.) Sponsors are defined using the following additional definition: Immediate events / reactions: Record real-time events to capture medically relevant, proactively administered systemic adverse events (including those related to the applied product) occurring within the first 30 minutes after vaccination.

[0428] Response to the request: The requested response is an “expected” adverse reaction (sign or symptom) observed and reported under the conditions (natural and onset) pre-listed in the protocol and CRB (e.g., fever and runny nose occurring between D0 and D28 post-vaccination).

[0429] By definition, a response to a request should be considered relevant to the product being applied.

[0430] For vaccines administered nasally, the requested response may be a requested site-of-application reaction or a requested systemic reaction.

[0431] Actively provided AE / AR: Actively provided adverse events (AEs) are observed AEs that do not meet the criteria pre-listed in the CRB regarding the diagnostic and / or post-vaccination morbidity window. For example, if fever between D0 and D28 is a requested response (i.e., pre-listed in the protocol and CRB), then fever starting on D28 is a requested response, while fever starting on D29 post-vaccination is an actively provided AE. Actively provided AEs include severe (SAE) and non-severe actively provided AEs.

[0432] AEs requiring medical attention (MAAE): MAAEs are new onsets or exacerbations of a condition that prompt a participant or their parent / guardian / legally authorized representative to seek unplanned medical advice at a physician's office or emergency room. Physician contacts made by phone or email are considered physician office visits for the purpose of MAAE collection. This definition does not include pre-planned medical office visits for routine medical care, or pediatric examinations or visits for chronic conditions that occurred prior to entry into the study. AEs identified during planned routine visits (e.g., upper respiratory tract infections, otitis media) are collected as MAAEs.

[0433] Reaction at application site: Application site reactions are irritants (ARs) at and around the application site (i.e., the nasal mucosa). They are believed to be related to the applied product.

[0434] Systemic AE: Systemic adverse events (AEs) refer to all AEs that are not related to the injection or application site. Therefore, they include systemic manifestations (such as headache, fever) as well as localized or localized manifestations that are unrelated to the vaccination or application site (e.g., localized conjunctivitis that does not occur at the application site).

[0435] Adverse events of special significance (AESI): Special attention should be paid to adverse events that are scientific and medical issues related to the sponsor's products or projects. Researchers should continuously monitor these events and communicate them promptly to the sponsor. Such events may require further investigation to characterize and understand them. Depending on the nature of the event, the research sponsor may also need to communicate rapidly with other parties, such as regulatory agencies. AESIs include serious (SAE) and non-serious, proactively provided AES.

[0436] The following definitions apply to different safety phases after vaccination: Acute phase: The acute phase begins with vaccination on day 0 and ends at midnight on day 28 (D28). The acute phase for the second vaccination begins on day 56 and ends at midnight on day 84. During the acute phase of the study, parents / guardians / legally authorized representatives can consult the study healthcare professional by telephone 24 hours a day regarding any illnesses that may occur during this period. Researchers will contact parents / guardians / legally authorized representatives daily for the first 7 days after each vaccination, and then three times a week from day 7 until day 28 after each vaccination. The 28-day safety follow-up acute phase coincides with the duration of attenuated live RSV virus shedding in RSV seronegative infants and young children. If a parent / guardian / legally authorized representative reports an SAE, a safety event that meets the study pause or termination criteria described herein, or symptoms suggestive of respiratory illness, a medical appointment should be arranged as described herein. During the acute phase, the eDC allows for daily safety monitoring of study participants and is programmed to alert parents / guardians / legally authorized representatives and the study site when symptoms suggestive of respiratory illness occur.

[0437] Post-acute phase: The acute-after phase for subjects receiving a single dose began at 12:01 a.m. on D29 and ended at midnight on D56. For subjects receiving two doses, the first acute-after phase began at 12:01 a.m. on D29 and ended at midnight on D56, unless the second dose was administered exactly on D56 immediately before the subject's first dose. The second acute-after phase for subjects receiving two doses began at 12:01 a.m. on D85 and ended at midnight on D112.

[0438] During the acute post-acute phase of the study, parents / guardians / legally authorized representatives are instructed to monitor their child and contact the researchers if their child develops symptoms suggestive of a serious adverse event. Medical attention should be scheduled if the parent / guardian / legally authorized representative reports an SAE or a safety event that meets the study suspension or termination criteria outlined herein.

[0439] Safety endpoint The primary endpoints for safety assessment are as follows (all infants and young children, regardless of baseline serum status): • Any actively administered systemic adverse events reported within 30 minutes of each vaccination.

[0440] • Requested application site reactions and systemic reactions (i.e., pre-listed in the subject's DC / eDC and CRB) occur within 28 days after each and any vaccination (i.e., the acute phase).

[0441] • Any proactively reported adverse events (AEs) occurring within 28 days of each and any vaccination (i.e., the acute phase). • Any occurrence of AESI within 28 days after each vaccination.

[0442] • Any MAAE occurring within 28 days after each vaccination.

[0443] • Any SAEs occurring throughout the study period.

[0444] • Other safety endpoints will be recorded or derived as described in the statistical analysis plan. Depending on the project, these may include the nature (preferred term in the MedDRA), time of onset, duration, number of days, intensity, relationship to vaccine, actions taken, whether the AE led to early termination of the study, severity, or outcome.

[0445] Safety assessment methods During each in-person or non-visit contact for vaccination, the investigator or representative either performs a focused physical examination (in-person visit) or asks the parent / guardian / legally authorized representative for responses to any requests and proactive adverse events (AEs) recorded on the diary card / electronic diary card, as well as any other AEs that may have occurred since the last visit. All relevant data are transcribed into the CRB according to the instructions provided by the sponsor.

[0446] This study monitored the safety, infectivity, replication, and immunogenicity of two doses of RSV ΔNS2 / Δ1313 / I1314L (Sanofi), with a focus on vaccine virus infectivity and replication 7 days after vaccination 1 and vaccination 2 (i.e., the percentage of virus shed by participants and the amount of vaccine virus titer in nasal swabs), which is the most quantifiable measure of the level of attenuation of the vaccine virus.

[0447] Immediate observation period after vaccination Subjects were observed for 30 minutes after each vaccination to ensure safety. Post-vaccination observations were recorded in the source file. Any adverse events (AEs) occurring during this period were noted in the source file and recorded in the CRB, as follows: • Actively provided whole-body AEs are recorded as immediate AEs in the CRB (existence is marked "yes" and details are collected).

[0448] • Requested and voluntary application site reactions, as well as requested systemic reactions, are recorded in the CRB in the same manner as any reaction that begins on the day of vaccination.

[0449] • SAEs are recorded in the CRB and reported to the sponsor in the same manner as any other SAE.

[0450] Reactivity (response requested from day 0 to day 28 after each vaccination) Following each vaccination, a DC / eDC and a digital thermometer will be provided to the subject's parents / guardians / legally authorized representative, along with instructions on their use. Subjects will record the following items on a diary / electronic diary card on the day of vaccination and for the next 28 days (i.e., day 0 to day 28) until the symptoms subside: • Daily temperature, and the method of administration. • Measure and record the intensity level of any requested site-specific reactions (e.g., runny nose) and systemic reactions (e.g., fever) daily.

[0451] • Measures taken for each event (e.g., medication). Use the following list in the CRB to categorize one or more actions taken by parents / guardians / legally authorized representatives in response to any requests for treatment and / or administration (all applicable items should be checked): • none • Medicine • Healthcare provider contact • Hospitalization • Suspension of research vaccination For the first 7 days after each vaccination, the parents / guardians / legally authorized representatives of the study subjects will be contacted by phone daily, and then three times a week after the D07 visit, to remind them to record all safety information in the diary card / electronic diary card.

[0452] If a call is made on a weekend or public holiday, it should be made on the next business day. If no contact is made by the designated date, the researcher will continue calling until contact is established. Each call attempt and its outcome is documented in the source document.

[0453] Tables 6 and 7 list the application site responses and intensity scales pre-listed in the diary card and CRB, respectively. * For measurable responses to nasal discharge and nasal obstruction, the parent / guardian / legally authorized representative determines whether medical intervention is needed and, if so, the type of intervention, and assigns it as a level 1 to 5 when reviewing the diary card. For all reactions other than fever, the parents / guardians / legally authorized representatives recorded the intensity level (level 1, 2, or 3) on a diary card. For fever, they recorded the body temperature and assigned it as level 1, 2, or 3 based on the information in the diary card.

[0454] Important considerations for accurate temperature assessment: Parents / guardians / legally authorized representatives should take the child's temperature once a day, ideally at the same time each day. The best time to take the temperature is in the evening when it is highest. Temperature should also be taken if any significant fever is present. Daily temperature readings and the method of measurement are recorded on a diary card, and the highest temperature is recorded in the CRB database. The preferred method of measurement in this study is rectal. Researchers also systematically collected pre-vaccination temperatures from the original files. Tympanic thermometers must not be used.

[0455] Proactively provided adverse events In addition to recording responses to requests, parents / guardians / legally authorized representatives are instructed to record any other medical events that may occur during the 28-day period following each vaccination. Space is provided in the journal card for this purpose.

[0456] Throughout the study, from enrollment to the last visit, information regarding SAEs was collected and assessed. Investigators reported any SAEs occurring at any time during the study period in the CRB according to the instructions provided by the sponsor; this included checking the “Serious” box on the AE CRF and completing the corresponding Safety Supplemental Information CRF. All information relating to SAEs was reported as part of the initial report or during follow-up reporting if relevant information became available later (e.g., outcomes, medical history, results of investigations, copies of hospital reports, and oral autopsy questionnaires, if used). In cases where a subject experienced a febrile seizure (a neurological event associated with fever and epilepsy), assessment was performed according to the “Febrile Seizure Case Definition and Collection Guidelines,” and the event was considered an SAE.

[0457] For each actively provided AE (whether severe or non-severe), the following information was recorded: • Start and Stop Dates (Stop dates for all relevant Actions that have been actively requested. For other events, the researcher will provide stop dates when available. Actions for which no stop date has been provided during the study are considered ongoing at the end of the study.) • Intensity of the event: a. Collect fever temperatures and analyze them according to the appropriate scale used for the response to the request.

[0458] b. All other actively provided AEs are classified according to the following intensity scale: i. Grade 1: A type of adverse event that is usually transient and may require minimal treatment or intervention. The event typically does not interfere with normal daily activities.

[0459] ii. Grade 2: A type of adverse event that can usually be mitigated by additional therapeutic intervention. This event interferes with daily living activities and causes discomfort, but does not pose a significant or permanent risk of harm to the study participant.

[0460] iii. Grade 3: A type of adverse event that disrupts daily living activities or significantly affects clinical status, or may require intensive therapeutic intervention.

[0461] • Is the AE (Adverse Event) related to the research product (for proactively provided systemic AEs)? As described in this article, researchers assess the causal relationship between the AE and the research product as either "unrelated" or "related".

[0462] • Measures taken for each AE (e.g., drug treatment) In CRB, actions taken by parents / guardians / legally authorized representatives for the treatment and / or management of any proactively presented adverse events are categorized using the following list (all applicable items should be checked): O None O drugs Contact healthcare providers O Hospitalization O Suspend research on vaccination • Whether AE is AESI, MAAE, and / or SAE.

[0463] For each SAE, the researchers completed all applicable severity criteria (outcome, time elapsed, and relation to the study procedure). • Did the adverse event (AE) lead to the suspension of the study? Adverse events requiring medical attention The same procedure as for other AEs was used to collect any MAAEs occurring within the first 28 days after vaccination (D0 to D28 for all subjects in cohorts 1, 2, 3, and 4, and D56 to D84 for subjects in cohorts 2 and 4). Acute respiratory illness requiring medical attention (MAARI) and acute lower respiratory illness requiring medical attention (MAALRI) events occurring within this time frame and outside the RSV surveillance season were classified as MAAEs.

[0464] Adverse events of particular concern (AESI) Use the same procedure as other AEs to collect any AESIs that occurred within the first 28 days after vaccination (D0 to D28 for all subjects in cohorts 1, 2, 3 and 4, and D56 to D84 for subjects in cohorts 2 and 4).

[0465] All adverse events of particular concern in this study will be graded according to the AIDS Division (DAIDS) Adult and Pediatric Adverse Event Severity Grading Scale, except for acute wheezing, which will be graded according to the Brighton Collaboration Wheezing Severity Grading System. The following adverse events of particular concern are derived from previous NIH research experience with similar candidate vaccines and were evaluated in this study: Acute otitis media • URI Pharyngitis • Cough without LRI • LRI • Wheezing • Loudsound • Rapid breathing • Acute wheezing • pneumonia • Laryngitis, tracheobronchitis Acute otitis media: Loss of tympanic membrane landmarks, accompanied by erythema and loss of mobility. This may or may not be related to fever or other respiratory symptoms. If possible, confirm the diagnosis with tympanometry. This diagnosis must be made by a medical professional.

[0466] pharyngitis: Pharyngeal erythema with exudate or pharyngeal erythema with swollen and tender lymph nodes. Note: This may be related to sore throat, painful or difficult swallowing. This diagnosis must be made by a medical professional.

[0467] Cough without LRI: Three or more coughs occurring within a 15-minute observation period over two or more consecutive days, or coughing that wakes the child from sleep. There should be no associated lower respiratory tract illness. This diagnosis must be made by a medical professional. Note: Not related to eating, drinking, or choking.

[0468] wheezing: A harsh, mid-pitched inspiratory sound associated with obstruction of the larynx or external trachea, often accompanied by a wheezing cough and hoarseness. This diagnosis must be made by a medical professional.

[0469] Luoyin: Abnormal lung sounds can be heard with a stethoscope. These sounds may be hissing (whistling), dry (crackling), or wet (sloshy), depending on the amount and density of fluid flowing back and forth in the airways. They must persist for at least 20 minutes and be evaluated and diagnosed by a medical professional, and confirmed by a second medical professional if possible.

[0470] Rapid breathing: An increased respiratory rate (>40 breaths per minute) is observed in infants aged 6-12 months, and in toddlers aged 1-3 years (>30 breaths per minute). This diagnosis must be made by a medical professional.

[0471] Acute wheezing: According to the Brighton Collaboration case definition, acute wheezing is a clinical sign for all levels of diagnostic certainty, as defined below: • Sudden onset, unexpected occurrence without warning, leading to significant changes in the subject's previously stable condition, and • Breath sounds can be heard during auscultation, and • It can be heard during exhalation, and the sound is mainly an exhalation sound, although the inspiratory component may appear in a different, more severe form than wheezing. Its characteristic is that it defines the following criteria for the level of diagnostic certainty: Level 1 • Based on the comparison between the digital stethoscope recording and the reference audio file, it was classified as wheezing, or • Wheezing is classified as such by two specifically trained healthcare providers (such as pulmonologists) or by formal auscultation training based on standard wheezing training tools. Level 2a (a specially trained healthcare provider) • Wheezing is classified as such by a healthcare provider (e.g., a pulmonologist) who has received specific training or formal auscultation training based on standard wheezing training tools, and • Immediate response to bronchodilator therapy, i.e., no wheezing or improvement in wheezing severity as recorded by a healthcare provider after treatment. Level 2b (2 healthcare providers) • The condition was classified as wheezing by two untrained healthcare providers (i.e., neither pulmonologists nor individuals with formal auscultation training based on standard wheezing training tools), and • Immediate response to bronchodilator therapy, i.e., no wheezing after treatment or improvement in wheezing severity as recorded by a healthcare provider, or • The infant was diagnosed with acute bronchiolitis Level 3 (Previous diagnosis) • Asthma is classified by a healthcare provider or caregiver who has not received specialized training (such as a parent / guardian / legally authorized representative), and • Previously diagnosed by a doctor with a respiratory disease characterized primarily by wheezing. The full Brighton Collaboration case definition is listed here, but level 3 diagnostic certainty will not be required for this study, and children pre-diagnosed with wheezing will not be enrolled in the study.

[0472] pneumonia: Rales and moist rales lasting more than 20 minutes, originating from the lower respiratory tract, are usually accompanied by tachypnea, and are less pronounced with coughing. This can be confirmed by X-ray showing areas of consolidation. If possible, clinical evaluation and diagnosis must be performed by a healthcare professional and confirmed by a second healthcare professional.

[0473] Laryngotracheobronchitis (croup): A barking cough, hoarseness, and inspiratory wheezing lasting more than 20 minutes should be assessed and diagnosed by a medical professional, and confirmed by a second medical professional if possible.

[0474] Table 8 lists the AESI DAIDS severity scale (excluding wheezing), ranging from level 1 to level 4. All deaths related to AESI are classified as Level 5.

[0475] Table 9 introduces the Brighton Collaboration wheezing severity grading system. I / E, Inhalation / Inhalation *Shortness of breath may even occur, but without other signs of respiratory distress. †Tachycardia may occur, but there are no other signs of respiratory distress. ‡ Or patients with a history of wheezing will have no sound in their chest upon auscultation. Causal relationship assessment Researchers assessed the causal relationship between each actively administered systemic adverse event (AE) and the applied product as either unrelated or related based on the following definitions: • Irrelevant – The adverse event (AE) is clearly / likely caused by other factors, such as underlying disease, treatment intervention, or concomitant therapy; or the delay between vaccination and the occurrence of the AE is incompatible with the causal relationship; or the AE began before the first vaccination. • Relevant – There is a “reasonable probability” that the AE was caused by the applied product, meaning there is evidence or argument to suggest a causal relationship. Note: By convention, all adverse events (AEs) reported at the application site (whether requested or offered) and all requested systemic AEs are considered to be related to the applied product and are therefore referred to as reactions, and no opinion from the investigator is required on the relevance.

[0476] Researchers follow up on any potentially product-related adverse events (whether severe or not) that persist at the end of the study until they completely disappear or the subject's condition stabilizes. Researchers inform sponsors of the date the event ultimately disappears or the date "chronic" is established.

[0477] Infectious There is no primary target for infectious diseases.

[0478] Immunity Definition of immunity RSV serum status In this Phase I / II study, serum IgA detection was chosen as a biomarker for RSV exposure in infants and young children. IgA serum status, i.e., those who have not been infected with RSV and those who have experienced RSV infection, was defined as undetectable or detectable serum anti-RSV A IgA antibodies, respectively.

[0479] RSV IgA serum status assessment The RSV IgA ELISA used to determine serum status will be performed by the sponsor or a qualified contract laboratory of the sponsor.

[0480] The methods used for assessing IgA serum status are summarized below.

[0481] RSV IgA Method Description IgA antibodies against RSV F antigen were measured using an anti-RSV F IgA ELISA. RSV F protein antigen was coated onto the surface of a microtiter plate. The plate was sealed, and unbound antigen was washed from the wells, with serially diluted human serum samples (test sample, reference, and mass control) incubated in the wells. Anti-RSV F protein-specific antibodies in the serum samples bound to the immobilized RSV F protein antigen. Unbound antibodies were washed from the wells, and a horseradish peroxidase (HRP)-conjugated goat anti-human IgA enzyme conjugate was added. The conjugate bound to the antigen-antibody complex. Excess conjugate was washed away, and a colorimetric substrate was added. The bound enzyme catalyzed a hydrolysis reaction, resulting in color development. The intensity of the color produced was proportional to the amount of antigen-specific IgA antibody bound in the well. The results were read on a spectrophotometer.

[0482] The concentration of IgA antibody against RSV F antigen was calculated in ELISA units (EU) / mL using six consecutive 2-fold dilutions, under specified conditions.

[0483] Immunogenic endpoint The primary endpoint for assessing immunogenicity is: • RSV A serum neutralizing antibody titers in subjects who have not been infected with RSV at D56 for cohorts 1, 2, 3 and 4 and at D84 for cohorts 2 and 4.

[0484] This article defines subjects who have experienced RSV and subjects who have not been infected with RSV.

[0485] Immunogenicity assessment methods The immunogenicity of candidate vaccines was assessed by measuring the titer of RSV A serum neutralizing antibodies (by micro-neutralization assay).

[0486] RSV neutralizing antibody assessment RSV micro-neutralization (MN) assays were performed by the sponsor or a qualified contracted laboratory of the sponsor.

[0487] The MN method used is summarized below. The development and qualification of this method have been completed; it will be validated prior to Phase III clinical trials.

[0488] RSV MN Method Description RSV neutralizing antibodies were measured using the MN assay. Serially 2-fold diluted test serum (pre-heat-inactivated) was mixed with a constant concentration of RSV-A2 strain (ATCC VR-1540). The mixture was inoculated into the wells of a 96-well microplate containing permissive HEp-2 cells (ATCCCCCL-23) and incubated for 2 days. Reduced viral infectivity (viral antigen production) due to antibody neutralization in the serum sample was detected by enzyme-linked immunosorbent assay (ELISA). RSV antigen production in cells was detected by sequential incubation with an RSV-specific mAb, a horseradish peroxidase anti-mouse IgG conjugate, and a chromogenic substrate after washing and fixation. The resulting optical density was measured using a microplate reader. A reduction in RSV infectivity compared to the virus control wells constituted a positive neutralization reaction, indicating the presence of neutralizing antibodies in the serum sample.

[0489] Secondary endpoints and evaluation methods Security Security Definition Security is defined as described above.

[0490] Safety endpoint The secondary endpoint of the security assessment is: • The titer of vaccine virus shedding in cohorts 1, 2, 3 and 4 on day 7 and in cohorts 2 and 4 on day 63 was measured by RT-PCR.

[0491] Security assessment methods The shedding of attenuated RSV vaccine strains in nasal swab samples was assessed using a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, which specifically detects and quantifies the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine strain in human nasal swab samples. Based on assessments of precision (intra-assay and intermediate precision), dilution accuracy, linearity, and specificity, the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) qRT-PCR assay is suitable for clinical testing of human nasal swab samples to support the development of RSV vaccine candidates.

[0492] RSV ΔNS2 / Δ1313 / I1314L (Sanofi) RT-qPCR method The RSV ΔNS2 / Δ1313 / I1314L (Sanofi) RT-qPCR method was performed at the sponsor's or a qualified contract laboratory of the sponsor.

[0493] The RSV ΔNS2 / Δ1313 / I1314L (Sanofi) RT-qPCR assay is used to measure viral shedding in nasal swab samples from research subjects. The development and identification of this method have been completed; it will be validated prior to Phase III clinical trials.

[0494] RSV ΔNS2 / Δ1313 / I1314L (Sanofi) RT-qPCR Method Instructions To quantify viral shedding in infants vaccinated with RSV ΔNS2 / Δ1313 / I1314L (Sanofi), a qRT-PCR assay was developed specifically to detect and quantify RSV ΔNS2 / Δ1313 / I1314L (Sanofi) in nasal swab samples.

[0495] The RSV ΔNS2 / Δ1313 / I1314L (Sanofi) qRT-PCR assay was designed using the Sigma LightCycler Probe system. This system comprises two hybridization probes designed to bind to a target 1–5 nucleotides apart. Probe 1 (donor) is labeled with a donor reporter gene at its 3' end. Probe 2 (recipient) is labeled with a recipient reporter gene at its 5' end. During the annealing step, the PCR primers and the LightCycler probe hybridize to their specific target regions, bringing the probes closer together. When this occurs, the donor dye is excited by the LightCycler, and energy is transferred from the donor to the recipient dye. The emission of the recipient reporter gene is detected by a photocycler at 640 nm.

[0496] If the probe binds but does not come close, no signal is generated. The Light Cycler probes used for qRT-PCR assays of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) target the deletion site of the NS2 gene in RSV ΔNS2 / Δ1313 / I1314L (Sanofi). Probe 1 binds before the deletion site, and probe 2 binds across the deletion site. Although the primers and probes may bind to wild-type RSV A due to high sequence similarity, the NS2 gene is over 500 nucleotides long, preventing the two probes from binding close enough to generate a signal, thus making this method highly specific.

[0497] Collecting nasal swab samples from infants can be difficult and the quality of the samples cannot be visually verified. Therefore, based on an assay developed by the CDC, the RNase P assay will be used in conjunction with the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) qRT-PCR assay to assess sample quality.

[0498] RNase P is a human housekeeping gene. Using an RNase P assay will limit the impact of false negatives due to improper sample collection or handling. Amplification in an RNase P assay indicates the presence of human cells in the sample. An RNase P Cp ≤ 37 indicates proper sample collection and preservation of sample integrity. An RNase P test is only performed when qRT-PCR (for detecting and quantifying the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine strain) produces a negative result.

[0499] Infectious Infectious endpoint The secondary endpoint for assessing infectivity is: • Infection with the vaccine virus in vaccine recipients. Infection is defined as a ≥ 4-fold increase in the titer of vaccine virus detected by RT-PCR in nasal swabs and / or in RSV A serum antibody titer or RSV serum anti-F IgG antibody titer.

[0500] Infectivity assessment methods The methods for assessing infectivity are as described above.

[0501] Immunogenicity Immunogenic endpoint The secondary endpoint for assessing immunogenicity is: • RSV serum neutralizing antibody titers in subjects who had experienced RSV at D56 for cohorts 1, 2, 3 and 4 and at D84 for cohorts 2 and 4.

[0502] • RSV serum anti-F IgG antibody titers for cohorts 1, 2, 3 and 4 at D56 and for cohorts 2 and 4 at D84.

[0503] • RSV A serum neutralization and serum anti-RSVF IgG antibody titer at least five months after the RSV season or the last vaccination.

[0504] Immunogenicity assessment methods As described above, subjects who have experienced RSV and subjects who have not been infected with RSV are defined.

[0505] The immunogenicity of candidate vaccines was assessed by measuring RSV F protein-binding antibody levels (by ELISA).

[0506] RSV anti-F IgG ELISA Anti-RSV F IgG ELISA measurements were performed at the sponsor's or a contracted laboratory qualified by the sponsor.

[0507] The ELISA methods used are summarized below.

[0508] RSV anti-F IgG ELISA method description Antibodies against the RSV F antigen were measured using an anti-RSV F IgG ELISA. In short, the RSV F antigen (from strain RSV A2) was coated onto a microtiter plate, and serial 2-fold dilutions of human serum samples were added and incubated to allow binding to the RSV F antigen. HRP-conjugated anti-human IgG detection antibodies were then added, followed by the colorimetric substrate. The concentration of IgG antibodies against the RSV F antigen was calculated using a qualified internal reference with assigned values ​​(International Units / mL) calibrated to WHO international standards (International Standard I for Respiratory Syncytial Virus Antiserum).

[0509] Data Management SAE Management During the study, SAE data (reported by the CRF regarding AEs, deaths, and safety supplemental information) were integrated into the sponsor's centralized GPV database upon receipt of these forms and after repeat checks. Each case was assigned a case identification number. Each case was assessed by the case management platform or its representatives and then reported to the relevant authorities as needed. The assessment of relevant cases was conducted in collaboration with global safety officials and the RMO. Follow-up information for completed cases was entered into the GPV database, and a new record for that case was created.

[0510] The information from cases in the GPV database is consistent with that in the clinical database.

[0511] Clinical and laboratory data management Clinical data (defined as all data reported in the CRB) and laboratory data are processed by the sponsor’s Clinical Data Management (CDM) platform or an authorized representative.

[0512] During the study, clinical data reported in the CRB were integrated into the clinical database managed by the sponsor's CDM platform. Quality control measures, in the form of site-specific data monitoring and computer-based logical and / or consistency checks, were systematically applied to detect errors or omissions. Furthermore, sponsor staff conducted multiple data reviews throughout the study. Any issues related to the reported clinical data were submitted to the investigator for resolution using the EDC system. Each step of this process was monitored through the implementation of individual passwords to maintain appropriate database access and ensure database integrity.

[0513] Validation of immunogenicity data was performed at the laboratory level according to laboratory procedures. Consistency of information from the laboratory was checked before integration into the clinical data warehouse.

[0514] After integrating all corrections in the complete dataset and after reconciling the SAE information available from the CDM and GPV departments, the database was published for statistical analysis.

[0515] Data review Before the database was locked, the data underwent a blind review process led by data management personnel.

[0516] Determination of statistical methods and sample size Statistical methods for main objectives This statistical method is based on using two 95% confidence intervals (CI).

[0517] The 95% confidence intervals for point estimates of the proportions are calculated using the exact binomial distribution (Clopper-Pearson method).

[0518] For immunogenicity data, assume the logarithm of titration / titer ratio. 10 The transformation follows a normal distribution. First, it uses standard computation for a normal distribution in logarithmic order. 10 Calculate the mean and 95% CI on (titration / titration ratio). Then apply an antilogarithmic transformation to the calculated results to calculate the geometric mean titration (GMT) and geometric mean titration ratio (GMTR) and their 95% CI.

[0519] Security Requested adverse events (ARs), proactively reported adverse events (including SAEs), major adverse events (MAAEs), and adverse events syndromes (AESIs) were summarized. Key parameters were described with a 95% confidence interval. In all subjects, regardless of baseline serological status, the vaccine group presented at least the following parameters after each and any vaccination: • An active-induced systemic adverse event (AE) occurs within 30 minutes of administration (immediately active AE). • Requested site-specific and systemic responses within 28 days after each and any administration, depending on the occurrence, timing, intensity, number of days, actions taken, and whether the response led to early termination of the study. The highest intensity will be used when more than one intensity level is reported over a period of time.

[0520] • Actively provided adverse events (AEs) occurring within 28 days after each and any administration, based on system organ class (SOC) and preferred terminology (PT), including their relationship, intensity, timing of onset, duration, and whether the AE led to early termination of the study.

[0521] • Based on SOC and PT, all SAEs that occurred throughout the study, severity criteria, timing of onset, outcomes, relationships, and whether the SAEs led to the early termination of the entire study. • Report all MAAEs and AESIs and their relationships within 28 days of each vaccination via SOC and PT. Posterior distribution-based Bayesian methods can be used to assess the differences between each RSV formulation and placebo on the following safety endpoints: • Any grade 3 lower respiratory tract disease such as wheezing, pneumonia, persistent tachypnea, laryngotracheobronchitis • Level 3 fever Immunogenicity For each vaccine group in subjects who have not been infected with RSV, point estimates and their 95% CIs are proposed for the RSV A neutralizing antibody titer at D56 for cohorts 1, 2, 3, and 4, and at D84 for cohorts 2 and 4: • GMT • GMTR based on baseline antibody titration • Serum response rate is defined as the percentage of subjects whose RSV A serum neutralizing antibody titer increases ≥4-fold from baseline.

[0522] • Shows the inverse cumulative distribution curve (RCDC) • Pairwise comparisons between vaccine groups can be performed as exploratory analyses to compare seroreactivity or GMT for descriptive purposes.

[0523] The 95% CI of the proportion difference between the two groups is calculated using the Wilson score method, without continuity correction. The CI of the GMT ratio between the two groups is calculated based on the logarithmic approximation of the normality between the two groups. 10 Calculation of the difference in average conversion titration amount.

[0524] Statistical methods for secondary objectives Statistical methods Security For each vaccine group, point estimates of the following parameters and their 95% CIs were derived from baseline serological status 7 days after each vaccination (D7 for cohorts 1, 2, 3, and 4, and D63 for cohorts 2 and 4): • Measuring GMT of vaccine virus shedding by RT-PCR Infectious For each vaccine group, point estimates and their 95% CIs for the following parameters are derived from baseline serological status after vaccination 1 (D56) for cohorts 1, 2, 3, and 4, and after vaccination 2 (D84) for cohorts 2 and 4: • The proportion of vaccine recipients infected with the vaccine virus. Infection was defined as a ≥ 4-fold increase in the amount of vaccine detected by polymerase chain reaction (PCR) in a nasal swab and / or in the titer of RSV A antibodies or RSV anti-F IgG antibodies in serum.

[0525] Immunogenicity Point estimates of GMT and GMTR and their 95% CIs are provided per vaccine group for the following endpoints: • RSV A serum titer and antibody titer in subjects who had experienced RSV at D56 for cohorts 1, 2, 3, and 4, and at D84 for cohorts 2 and 4. • RSV serum anti-F IgG antibody titers based on baseline serum status at D56 for cohorts 1, 2, 3 and 4, and at D84 for cohorts 2 and 4.

[0526] • RSV A serum neutralization and anti-RSV F IgG antibody titer based on baseline serum status at least 5 months after the RSV season or the last vaccination.

[0527] Serum responses were also shown, if applicable.

[0528] Analysis set Complete analysis set The complete analysis set (FAS) was defined as a subset of randomized subjects who had received at least one dose of the study vaccine. Data from any subject who was laboratory-confirmed to be infected with wtRSV from the date of wtRSV infection were excluded from the immunological and viral shedding analyses.

[0529] Security Analysis Set The Safety Analysis Set (SafAS) was defined as subjects who had received at least one dose of the study vaccine. All subjects underwent safety analyses after each dose based on the specific vaccine they received, and after any subsequent dose based on the vaccine they received at the time of their first administration.

[0530] Safety data from vaccine records received according to the protocol design were excluded from the analysis (and listed separately).

[0531] Analysis set by scheme The per-protocol analysis set (PPAS) is a subset of the FAS. Two specific PPAS are defined: PPAS1 after one administration (for subjects in cohorts 1, 2, 3, and 4) and PPAS2 after two administrations (for subjects in cohorts 2 and 4).

[0532] Participants exhibiting at least one of the following protocol biases were excluded from the PPAS: • No baseline serological blood sample was collected during the first visit (D0).

[0533] • Subjects with temporary contraindications did not receive the vaccine within the appropriate time window after randomization.1

[0534] • Subjects did not meet all of the inclusion criteria specified in the protocol or met at least one of the exclusion criteria specified in the protocol. • Subjects who have not received a vaccine / have not completed their vaccination schedule • The subject received a vaccine other than the one randomly assigned to him / her. • Vaccines were not prepared and / or administered according to the protocol. • Subjects received a protocol-prohibited therapy prior to the collection of post-vaccination serological blood samples. Subjects were diagnosed with wild-type (wt) RSV prior to the collection of post-vaccination serological blood samples.

[0535] • Subjects whose unblinding was performed by researchers in an emergency.

[0536] Subjects exhibiting at least one of the following protocol biases were excluded from PPAS1 only: • No serological blood sample was collected at the third visit after vaccination. • Serological blood samples were not collected within the appropriate time window at the third medical visit 1 day after vaccination. Subjects exhibiting at least one of the following related protocol biases were excluded from PPAS2 only (for subjects in cohorts 2 and 4): • The subject did not receive the vaccine within the appropriate time window at the third visit. • No serological blood sample was collected at the 5th visit 2 days after vaccination. • Serological blood samples were not collected within the appropriate time window at the 5th visit 2 days after vaccination. In addition to the reasons listed above, subjects are also excluded from PPAS if their baseline serological sample or their post-vaccination serological sample does not produce valid test results (i.e., lack of RSV A serum neutralizing antibody titer results).

[0537] Note: For PPAS1 (after 1 administration), the time points considered are: for vaccination, D0 (Visit 01), and for serological samples after vaccination, D56 (Visit 03). For PPAS2 (after 2 administrations), the time points considered are: for vaccination, D0 (Visit 01) and D56 (Visit 03), and for serological samples after vaccination, D84 (Visit 05).

[0538] Other analysis sets Randomized subjects A randomized subject is a subject to whom a particular vaccine group has been assigned.

[0539] Populations used in the analysis Safety analysis was performed on SafAS. Subjects were analyzed according to the vaccine they actually received after each vaccination and according to the vaccine received at the first administration after any vaccination.

[0540] Immunogenicity analysis was performed on the full analysis set, and the primary immunogenicity parameters were performed on the per-protocol analysis set. In FAS, subjects were analyzed according to the randomly assigned vaccine group. In PPAS, subjects were analyzed according to the vaccine they actually received.

[0541] Handling of missing data and outliers Safety No imputation was performed. However, in the statistical analysis, missing relationships were considered relevant. No outlier search was performed. In all subject listings, partial and missing data were clearly marked as missing.

[0542] Immunogenicity No imputation of missing data was performed. No outlier tests or searches were performed.

[0543] To calculate the GMT and the proportion of subjects with nAb titers above the threshold, any pre-vaccination or post-vaccination value reported as < lower limit of quantification (LLOQ) was converted to a value of ½ LLOQ.

[0544] For the calculation of GMTR, when only the numerator or denominator < LLOQ, any pre-vaccination value reported as < LLOQ was converted to LLOQ, and any post-vaccination value reported as < LLOQ was converted to a value of ½ LLOQ. If both the numerator and denominator were < LLOQ, both were converted in the same way so that the individual titer ratio = 1.

[0545] Any value reported as > upper limit of quantification (ULOQ) was converted to ULOQ.

[0546] Efficacy Missing data was not estimated. No outlier testing or search was performed.

[0547] Mid-term / Preliminary Analysis A step-by-step approach was used for analysis.

[0548] Several blinded early safety data reviews were conducted on safety data collected from subjects in each cohort at specific time points.

[0549] The following describes an unblinded interim analysis of subjects in cohorts 1, 2, and 3, and at least 90 enrolled subjects in cohort 4. The interim analysis involved subjects for whom safety data up to the D84 time point and who had D84 immunization results were available. Specific procedures were implemented to maintain blinding at both the subject and investigator levels.

[0550] A non-blinded early analysis is planned for participants in all cohorts (cohorts 1, 2, 3, and 4). The early analysis will proceed when all participants have provided safety data up to the D84 time point and D84 immunogenicity results are available. This early analysis requires unblinded data; specific procedures will be implemented to maintain blinding at both the participant and investigator levels. Based on the results of this analysis, the dosage for future studies will be determined.

[0551] The final unblinded statistical analysis will address the objectives for all subjects (cohorts 1, 2, 3, and 4), including post-season data.

[0552] No statistical adjustment is required because no hypotheses will be tested.

[0553] Determination of sample size and statistical power Since there were no statistical hypotheses in this study, the sample size was not calculated.

[0554] The plan is to sequentially assign the total 300 participants to one of four cohorts: • Cohort 1 (single administration): 40 subjects, i.e., 20 subjects in each vaccine group (low dose of RSV or placebo). • Cohort 2 (2 administrations): 40 participants, i.e., 20 participants in each vaccine group (low-dose RSV or placebo). • Cohort 3 (single administration): 40 subjects, i.e., 20 subjects in each vaccine group (high dose of RSV or placebo). • Cohort 4 (2 administrations): 180 subjects, i.e., 60 subjects in each vaccine group (high dose RSV or low dose RSV or placebo).

[0555] • The actual number of participants enrolled in each group will be discussed in the interim results below.

[0556] Although no statistically strong hypotheses were made or sample size was calculated in this study, the sample size of 100 subjects in the low-dose RSV group (20 from cohort 1, 20 from cohort 2, and 60 from cohort 4) provided a 95% probability of observing an event with a true incidence of 3%. The sample size of 80 subjects in the high-dose RSV group (20 from cohort 3 and 60 from cohort 4) provided a 95% probability of observing an event with a true incidence of 3.75%.

[0557] This study plans to enroll a total of 300 participants. This equates to 120 participants in the placebo group, 100 participants in the low-dose RSV group, and 80 participants in the high-dose RSV group. Table 10 below presents the total number of participants based on serological status and RSV group, according to different possible RSV experience rates ranging from 5% to 35% (derived from LID / NIH screening data), and provides an overall overview of the proportion of participants enrolled in the study who have not been infected with RSV / have experienced RSV.

[0558] [Table 10]: Number of subjects divided by serological status, total number, and RSV group, based on the possible RSV infection rate ranging from 5% to 35%. Example 2: Interim Results of the VAD00001 Study In the VAD00001 study, approximately 155 study participants (infants and young children) received the investigational product at any dose or dosing regimen. The final number of investigational product recipients is unknown as the study has not yet been fully unblinded. Interim results from VAD00001 showed that the candidate vaccine was well-tolerated, exhibiting high infectivity, genetic stability, and a robust immune response.

[0559] The ongoing Phase I / II trial (VAD00001) is being conducted in the United States and South America (Chile and Honduras). In this trial, two dose levels (5.6 log) of the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine are being compared. 10 PFU and 6.2 log 10 The safety, immunogenicity, and infectivity of PFU have been and will continue to be evaluated in a continuous cohort.

[0560] This study was conducted to ensure a safe, stepwise approach was used to achieve the doses in cohort 4. Cohort 4 was designed to use 5.6 log... 10 PFU / dose and 6.2 log 10Both PFU / dose were used to 1) confirm, as indicated by data from NIH trials, 6.2 log 10 PFU / dose can be used in Phase III (with 5.6 log). 10 (PFU / dose reduction), and 2) explore the acceptability of the safety, infectivity and immunogenicity results of the two doses tested to clinically demonstrate the future dose range boundaries for the non-frozen liquid formulation for eventual commercialization.

[0561] Study participants aged 6–18 months have been and will continue to be assigned to receive the following interventions: Cohort 1 (planned 40 participants, 36 enrolled): 5.6 log1000 doses administered once. 10 PFU RSV ΔNS2 / Δ1313 / I1314L (Sanofi) or placebo Cohort 2 (planned 40 participants, 21 enrolled): 2 applications of 5.6 log₂5. 10 PFU RSV ΔNS2 / Δ1313 / I1314L (Sanofi) or placebo, 2 months apart. Cohort 3 (planned 40 participants, 22 enrolled): 6.2 log1000 doses administered once. 10 PFU RSV ΔNS2 / Δ1313 / I1314L (Sanofi) or placebo Queue 4 (planned 180 participants, 180 participants enrolled): 2 applications of 5.6 log₂. 10 PFU RSV ΔNS2 / Δ1313 / I1314L (Sanofi), 6.2 log 10 PFU RSV ΔNS2 / Δ1313 / I1314L (Sanofi) or placebo, 2 months apart. Baseline RSV serum status: R+ / R- (the definition used in the study is below).

[0562] Blood samples were collected from cohorts 1 and 3 before and 56 days after vaccination. Blood samples were collected from cohorts 2 and 4 before each dose and 28 days after the second dose. All participants in cohorts 1, 2, 3, and 4 provided blood samples one month after the end of the RSV season or at least five months after the last vaccination to measure post-season RSV antibody titers. This was to determine whether a four-fold or greater increase in RSV antibody titers had occurred during the RSV season, indicating infection with wild-type RSV but undetectable by surveillance, and to investigate residual antibody titers one RSV season later.

[0563] Nasal swab samples have been and will continue to be collected from all participants 7 days after the first vaccination for cohorts 1, 2, 3, and 4; and 7 days after the second vaccination for cohorts 2 and 4. These samples have been and will continue to be used for testing for vaccine shedding and respiratory pathogen infection. All study participants will continue to provide nasal swabs when reporting illness and for 48 hours thereafter.

[0564] Safety follow-up included immediate monitoring 30 minutes after vaccination, collection of requested adverse reactions, proactively provided AEs, MAAEs, and AESIs within 28 days after each vaccination, and SAEs throughout the trial (up to 12 months).

[0565] As planned, a non-blinded interim analysis of dose selection for the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine was conducted on participants from cohorts 1, 2, and 3, as well as the first 91 participants enrolled in cohort 4. The interim analysis has been and will continue after participants provided safety data up to 28 days after the second vaccination and after immunization results were obtained at that time. Future dose selection (i.e., 6.2 log...) will be developed. 10 PFU (target midpoint for future operations) has been based on a descriptive comparison of the safety, infectivity, and immunogenicity of either dose in all participants up to 28 days after the second vaccination. The inclusion of either dose in future operations was assessed. The results of the interim analysis are summarized below.

[0566] Infants with detectable serum IgA against RSV F at baseline were considered to have experienced RSV, while infants without detectable serum IgA against RSV F at baseline were considered to have not been infected with RSV. This approach also allows for the differentiation between participants who have experienced RSV and those who have not (who have had placental transfer of serum antibodies), serving as a more discriminative assay. The fundamental principle behind explicitly defining participants who have not been infected with RSV and those who have experienced RSV in Phase I / II studies is to thoroughly assess the safety of the live attenuated vaccine (LAV) in uninfected populations, as previous RSV infection can affect the infectivity of subsequent RSV infections, including LAV. Given that the IgA test is a more discriminative assay, results are presented based on an assessment of the participants' serological status at baseline using this serum IgA assay against RSV F, as follows: R-; Participants who have not been infected with RSV: titer < limit of detection [LOD] R+; Participants who have experienced RSV: titration ≥ LOD Other situations are undetermined. The interim results presented below include 167 participants who underwent safety assessments (50.3% female, mean age 11.0 ± 3.86 months, and 5.4% Black / African American, 38.3% Hispanic / Latino) and 139 participants who underwent infectivity and immunogenicity assessments (for whom IgA serological status data were available). Of the 170 participants recruited at the time of this interim analysis, 3 did not receive the study intervention and were therefore not included. Participants with documented RSV respiratory illness prior to the assessment time point were excluded from the immunogenicity analysis at the corresponding time point.

[0567] Safety, immunogenicity, and infectivity conclusions Security The safety profile of the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine was evaluated in all infants and young children after each and any single administration, regardless of baseline serological status. Post-vaccination reported symptoms were comparable between groups and cohorts. In this moderate-sized group of participants at the interim analysis, there was a trend toward requested application site reactions observed in a higher proportion of participants in the high-dose group after vaccination 1, while AESIs following any single vaccination showed a trend toward being more common in the low-dose group.

[0568] Two unrelated SAEs (outside the acute phase) were reported during the study: one grade 3 case in the low-dose group (left hand fat pad) and one grade 2 case in the placebo group (RSV-MAARI). Two AESIs in each of the low-dose group (nasopharyngitis), high-dose group (croup and wheezing in the same participant), and placebo group (nasopharyngitis and cough) after any vaccination were classified as related by the investigators. All VAD00001 study participants will continue to be visited for one RSV season prior to the start of the Phase III program. Swab data collected during disease visits are still anticipated and will be provided in subsequent analyses. Given the impact of the Covid-19 pandemic on RSV circulation during participation in cohorts 1, 2, and 3, laboratory-confirmed disease rates in these cohorts may be lower than in cohort 4. While we do not expect this candidate to enhance disease due to the attenuated nature and the available data to date, we will comment on available data regarding RSV disease following vaccination (when this becomes available).

[0569] Overall safety was comparable between participants who had not been infected with RSV and those who had experienced RSV infection.

[0570] As of November 30, 2022, no deaths had been reported in the study.

[0571] Overall, the safety data indicate an acceptable safety profile for the candidate at both dose levels (see Tables 11 and 12: Safety Overview after Vaccination 2 – Safety Analysis Set).

[0572] Immunogenicity Compared to 61% and 47% of RSV-naïve participants in the low-dose and high-dose groups, respectively, after one dose of vaccine, approximately 70% (70%) of vaccine recipients in both the low-dose and high-dose groups achieved a 4-fold response in neutralizing antibody titers after a second dose. This increase in the percentage of participants achieving a 4-fold response after a second administration supports the use of a second dose in this population. A 70% increase in this fold after a second administration is consistent with the candidate vaccine's expected clinical efficacy target of 70%. After one or two vaccinations, 75% of people experienced a 4-fold increase in serum neutralizing antibody titers. The percentage of RSV-naïve participants who also achieved a 4-fold response in neutralizing antibody titers after one vaccination (36% in the low-dose group and 22% in the high-dose group) suggests a potential benefit for this subgroup as well. It must be noted that this is from an interim analysis of a moderate sample of participants at this time (n = 20 out of 97 vaccine recipients).

[0573] In summary, these data strongly support the use of the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine in operational range studies against the candidate, including doses at 5.6 log PFU / dose and 6.2 log PFU / dose. (See Tables 13 and 14.) Vaccine virus shedding and infectivity Following each vaccination, vaccine virus shedding was considered as a factor of vaccine infectivity, in addition to a fold increase in neutralizing antibody titers or serum IgG levels. High levels of vaccine infectivity (over 80% and 70% of RSV-uninfected participants, respectively, after the first and second vaccinations) support promising vaccine candidates associated with good vaccine infectivity. When infectivity was considered after either vaccination, over 90% of participants had evidence of infection. Relatively high infectivity was found in a small cohort of RSV-experienced participants (80% and 33.3% in low- and high-dose recipients after a single vaccination, and 60% and 50% after a second vaccination), suggesting promising prospects for this group. Furthermore, the significant decrease in the percentage of vaccine virus shedding (approximately 20%) in RSV-uninfected participants after the second vaccination, compared to over 70% after the first vaccination (as previously documented with other effective attenuated live mucosal virus vaccines), is characteristic of subsequent “challenges” in the form of a second vaccine dose, which are characterized by a significant reduction in vaccine virus shedding. It is noteworthy that the available shedding data for this cohort comes from data at a single time point following each vaccination (seven days post-vaccination). While this is consistent with the peak viral shedding points documented in other RSV live attenuated vaccine (LAV) trials, some shedding may have been missed. This limitation in available shedding data makes the results particularly encouraging. (See Tables 15 and 16.) Overall Conclusion Interim analysis results showed that the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) candidate vaccine has promising safety, immunogenicity and infectivity.

[0574] No safety issues were found after administration of one or two doses of the investigational RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine at any dose level or based on baseline serological status.

[0575] The conclusions regarding vaccine viral shedding and immunity based on baseline IgA serological status demonstrated significant vaccination at both dose levels, and among RSV-naïve participants, 70% of RSV-naïve vaccine recipients achieved a 4-fold response in serological neutralizing antibody response after the second dose at both dose levels.

[0576] The results support an operational range for the candidate, which includes the range at 5.6 log. 10 PFU / dosage and 6.2 log 10 The dosage of PFU / dose will be determined, and support will be provided for the future development of the candidate. Early analysis will be conducted on day 28 after all participants have completed vaccination, provided all results are unblinded. Example 3: Immunogenicity and safety study of three different dose concentrations of respiratory syncytial virus (RSV) vaccines in infants and young children. Project Title: This parallel-group, phase III randomized, observer-blinded, placebo-controlled, multicenter, multinational, multigroup study aims to demonstrate the non-inferiority of the low-dose immune response compared to the standard dose and to assess the safety of the respiratory syncytial virus vaccine in infants and young children.

[0577] reason: To supplement the stability study of the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine within its shelf life, this study will investigate the immunogenicity and safety of three vaccine dose concentrations (i.e., low [LD: target 5.4 log10 PFU / dose], standard [SD: target 6.4 log10 PFU / dose], and high [HD: target 7.0 log10 PFU / dose]). Therefore, the aim of the study is to assess whether the LD, as assessed by RSV A and RSV B serum neutralizing antibodies, is non-inferior to the SD at 28 days after the second vaccination (day 85). The goal is to demonstrate that the immune response induced by the LD is not inferior to that of the SD. Furthermore, the study aims to investigate the safety of the vaccine in infants and young children born at term (i.e., ≥ 37 weeks of gestation) and preterm (i.e., 28 to 36 weeks of gestation). Inclusion / Exclusion Criteria Inclusion criteria Participants are eligible to participate in the study only if they meet all of the following criteria: age The age at the date of inclusion was 6 months to <22 months ("6 months to <22 months" means from the day of the participant's 6-month birthday to the day before their 22-month birthday. The second vaccination should be administered before the participant reaches 24 months of age.) Participant types and disease characteristics Healthy participants are identified through medical evaluation (including medical history).

[0578] For cohort 1 and cohort 2 (subject to satisfactory safety profiles of the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine in cohort 1): Participants were born between 28 and 36 weeks of gestation and were assessed by the investigators as medically stable, defined as: "medically stable" means that the condition of preterm infants does not require extensive medical support or ongoing management of debilitating illness and that they have demonstrated a clinical process of sustained recovery at the time they received the first dose of the study intervention.

[0579] For queue 2: Participants were born at full term (≥ 37 weeks of gestation). Informed consent I01. Informed consent has been signed and dated by the parent or other LAR (or by an independent witness if required by local regulations). Other inclusion cases Participants and their parents / LARs were able to attend all scheduled appointments and comply with all study procedures.

[0580] Exclusion criteria Participants are ineligible to participate in the study if they meet any of the following criteria: Medical conditions E01. Known or suspected congenital or acquired immunodeficiency; or having received immunosuppressive therapy, such as chemotherapy or radiotherapy for cancer, within the past 6 months; or having undergone long-term systemic corticosteroid therapy (using prednisone or its equivalent for more than 2 consecutive weeks within the past 3 months). E02. Known systemic allergic reaction to any component of the study intervention, or a history of life-threatening reactions to the study intervention used in the study or to products containing any of the same substances (the components of the study intervention are listed elsewhere). E03. Patients with chronic diseases that the researchers believe may interfere with the progress or completion of the study (chronic diseases may include, but are not limited to, heart disease, lung disease (including any history of reactive airway disease or treatment with bronchodilators or inhaled steroids), atopic diseases, kidney disease, autoimmune diseases, diabetes, mental illness, and known congenital or hereditary diseases). E04. Medically diagnosed history of wheezing (children with a history of recurrent wheezing will be excluded. Children who have had a single episode of wheezing may be included if the wheezing episode is unrelated to hospitalization or if there is no family history of wheezing).

[0581] E05. Any acute febrile illness within the past 48 hours that, in the investigator's opinion, is severe enough to interfere with successful vaccination on the day of vaccination. Potential participants should not be included in the study until the symptoms subside or the febrile event diminishes.

[0582] E06. Cases of persistent viral respiratory infection (including COVID-19, influenza, rhinovirus, etc.) that may have been diagnosed at enrollment. Potential participants should not be included in the study until the respiratory infection has subsided.

[0583] E07. Members of families containing immunocompromised individuals, including but not limited to: • People infected with human immunodeficiency virus (HIV) • Individuals who received chemotherapy within the 12 months prior to study enrollment • People who have received (within the past 6 months) or are currently receiving (at enrollment) immunosuppressants • People who receive solid organ or bone marrow transplants According to researchers, individuals may come into close contact with other immunocompromised individuals within 30 days of each vaccination.

[0584] Previous / companion therapy E08. The participant’s mother received or planned to receive the investigational RSV vaccine during pregnancy and / or breastfeeding.

[0585] E09. Received or planned to receive any of the following vaccines before enrollment or after the first study intervention: • The first study involved any other intranasal attenuated live vaccine administered within 28 days before or after the intervention.

[0586] • Unless administered on the day of the first study intervention, the individual must not have received any other injectable live attenuated vaccine within 28 days prior to or after the intervention. Concurrent administration on the day of the first study intervention is permitted.

[0587] E10. At enrollment, the candidate had received an investigational RSV vaccine or any anti-RSV product (such as ribavirin or RSV immunoglobulin). The candidate had received nirsevimab within 6 months prior to the first administration of the investigational vaccine, or palizumab within 3 months prior.

[0588] E11. Received immunoglobulin, blood, or blood-derived products within the past 3 months.

[0589] E12. Intranasal and intraocular medications were administered within 3 days prior to study enrollment. Previous / accompanying clinical research experience E13. At the time of enrollment in the study, or planning to participate in another investigational clinical study of a vaccine, drug, medical device, or medical procedure during the current study period.

[0590] Other exclusion scenarios E14. Deprivation of liberty in emergency situations or during involuntary hospitalization.

[0591] E15. The biological or adopted child of a researcher or employee who is directly involved in the proposed study.

[0592] Brief summary: This study will be a phase III parallel-group, randomized, observer-blinded, placebo-controlled, multinational, multicenter, multigroup study involving 947 healthy children enrolled at 6 months of age. The aim is to evaluate the non-inferiority of the immune response to LD compared to the SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine, and the safety of the SD and HD vaccines administered intranasally and compared to placebo in preterm infants and in full-term infants.

[0593] Detailed research information includes: The study duration for each participant is approximately 8–9 months, including a safety follow-up telephone call 6 months after the second study intervention was administered.

[0594] The study intervention will be administered to participants on D01 and D57 (one intranasal administration per nostril at each time point).

[0595] The frequency of visits is shown in Table 1. Safety data will be collected during all study visits and at designated time points via contact with the study participants' parents / legally acceptable representatives (LARs).

[0596] Number of participants: A total of 947 participants are expected to be randomized: 35 in queue 1 and 912 in queue 2.

[0597] Research group and duration: In each study group (RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine group and placebo group), eligible participants were randomized to receive either a dose of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine or placebo twice via intranasal administration (56 days apart, i.e., on D01 and D57).

[0598] The research group is as follows: Enrollment of participants in Cohort 1 (preterm birth) will be conducted sequentially / stepwise. Step 1: The first 20 participants will be randomized in a 1:1 ratio to receive SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) or placebo. Step 2: Based on the acceptability of the safety profile (up to 28 days after the first vaccination), an additional 15 participants will be randomized in a 2:1 ratio to receive HD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) or placebo.

[0599] Step 1: • Group 1 (N=10): Two administrations of SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) (target 6.4 log10 PFU / dose) • Group 2 (N=10): Two doses of placebo According to the safety specifications of SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi): Step 2: • Group 3 (N=10): Two administrations of HD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) (target 7.0 log10 PFU / dose) • Group 4 (N=5): Two doses of placebo Based on the acceptable safety profile of HD in Cohort 1, enrollment in Cohort 2 will be open to preterm infant participants.

[0600] Participants in queue 2 will be randomly assigned to groups 1 through 4 in a ratio of 2:2:1:1.

[0601] Group 1 (N=304): Two administrations of LD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) (target 5.4 log10 PFU / dose) Group 2 (N=304): Two administrations of SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) (target 6.4 log10 PFU / dose) Group 3 (N=152): Two administrations of HD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) (target 7.0 log10 PFU / dose) Group 4 (N=152): Two doses of placebo Queues 1 and 2 will be grouped in parallel.

[0602] At each vaccination visit, 0.1 mL of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine or placebo will be administered into each nostril using the study device.

[0603] The study period for each participant is approximately 8-9 months.

[0604] Research intervention: Study Drug 1: LD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine (target 5.4 [5.6 ± 0.3] log10 PFU / 0.2 mL) • Form: Nasal spray liquid • Composition: Each 0.2 mL dose of RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine will contain a live attenuated RSV with i) a 523-nucleotide deletion in the NS2 gene, ii) an amino acid deletion in the L protein (Δ1313; deletion of S1313), and iii) a genetically stable mutation in the L gene (I1314L), to be delivered as a fine droplet mist using an intranasal nebulizer device at approximately 0.1 mL / nostril.

[0605] • Route of administration: Intranasal Study Drug 2: SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine (target dose 6.4 log10 PFU / 0.2 mL) • The dosage form, composition and route of administration are the same as those of Product 1.

[0606] Study Drug 3: HD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine (target 7.0 [6.9 ± 0.3] log10 PFU / 0.2 mL) • The dosage form, composition and route of administration are the same as those of Product 1.

[0607] Study Drug 4: Placebo • Form: Nasal spray liquid • Composition: Buffer solution, the same histidine-based formulation buffer used with RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine, approximately 0.1 mL delivered per nasal cavity. • Route of administration: Intranasal Statistical considerations: Statistical Hypothesis Main immunogenic targets Twenty-eight days after the second administration of the RSV ΔNS2 / Δ1313 / I1314L (Sanofi) vaccine to participants in cohort 2, the geometric mean titer (GMT) of neutralizing RSV (A and B) antibodies in the LD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) group will be compared with that in the SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) group. The following hypothesis will be tested for each RSV antibody: GMT(rsv, LD) and GMT(rsv, SD) are the GMTs of RSV antibodies (A and B) from LD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) and SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi), respectively, in cohort 2.

[0608] If the lower limit of the 2-sided 95% CI for the ratio of GMT between the LD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) group and the SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) group is >2 / 3 for both neutralizing RSV antibodies (A and B), then the null hypothesis of inferiority will be rejected, and non-inferiority (NI) of LD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) compared to SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) will be demonstrated.

[0609] Statistical analysis The study intervention group will summarize all endpoints at each time point. For the analysis of cohort 1, groups 2 and 4 (i.e., the placebo group) can be combined. Results for each cohort will be presented separately.

[0610] Generally, categorical variables will be summarized and presented using frequency counts, percentages, and CIs. The 95% CI of point estimates will be calculated using a normal approximation of the quantitative data and an exact binomial distribution of the percentages (Clopper-Pearson method). For GMT and GMTR, the 95% CI of point estimates will be calculated using a normal approximation, assuming they follow a log10 normal distribution.

[0611] Immunogenicity analysis will be performed on the protocol-as-a-set (PPAS) and can be validated on the full set (FAS), provided that the difference between the number of participants in the PPAS and the number of participants in the FAS is not less than 10%.

[0612] Security analysis will be performed on the Security Analysis Set (SafAS).

[0613] Primary endpoint Immunogenicity analysis Assuming the log10 transformation of the titration follows a normal distribution, the two-sided 95% CI of the GMT ratio between the LD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) group and the SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) group from cohort 2 will be calculated. The null hypothesis of inferiority will be rejected if the lower limit of the two-sided 95% CI is greater than 2 / 3 for both RSV A and RSV B antibodies.

[0614] Security Analysis Safety parameters (including the frequency of immediate response, requested response, proactively provided AE, MAAE, AESI, and SAE) will be described by the study intervention group after each and any study intervention administration, with the 95% CI of point estimates calculated using an exact binomial distribution of proportions (Clopper-Pearson method).

[0615] Mid-term analysis A preliminary, unblinded analysis will be performed on data collected up to day 28 following dose 2 to determine immunogenicity and safety for each cohort. Randomization coding will be made public to the sponsors, but will remain blinded at the location and participant / parent levels.

[0616] Sample size determination Cohort 1: This will include 35 preterm participants. This is an arbitrary sample size for generating preterm infant safety data. No statistical hypothesis testing will be performed on this cohort.

[0617] Cohort 2: A total of 912 participants will be enrolled, with 304 in each of the LD and SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) groups, and 152 in each of the HD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) and placebo groups. The allocation ratio is 2:2:1:1.

[0618] For the main immunogenic targets Using 243 evaluable participants in both the LD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) and SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi), this study declared non-inferiority to the primary immunogenic target based on a 2.5% one-sided alpha having approximately 90% efficacy. Taking into account an estimated 20% dropout rate, 304 participants will be enrolled in each of the LD and SD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) groups.

[0619] For the main security objectives The sample size of 152 participants in the HD RSV ΔNS2 / Δ1313 / I1314L (Sanofi) group will provide a 95% probability of observing an event with a 2% true incidence and a 78% probability of observing an event with a 1% true incidence.

[0620] Example 4: Intranasal Nebulizer Delivery Device Intranasal nebulizer delivery devices used to deliver RSV vaccines and placebos were tested to measure dose accuracy, spray droplet size distribution (DSD), spray plume geometry (PG), spray pattern (SP), and dose of infection titer.

[0621] Materials and methods Material Vaccine buffer; store at 2°C to 8°C; avoid light exposure.

[0622] The amplified high-dose DP batch #19-035-FBP / FP and the low-dose DP batch #19-054-FBP / FP are both stored at <-60°C.

[0623] MAD130 device, intranasal nebulizer, with 1 mL syringe and vial adapter (manufactured by Teleflex), batch #73J1700328.

[0624] Poly(lactic acid) plastic 3D printed dosing dispenser; 0.1 mL, blue (in-house manufactured).

[0625] RSV vaccine purified in bulk.

[0626] method The methods used in this article comply with FDA and EMA guidelines (US Food and Drug Administration. Industry guidance: Nasal sprays and inhalation solutions, suspensions and aerosols pharmaceuticals - chemistry, manufacturing and control documentation. Fed. Regist. 2002, 1-49. European Medicines Agency. Guideline on the Pharmaceutical Quality of Inhalation and Nasal Products; European Medicines Agency: London, UK, 2006; pp. 1-27).

[0627] Injection weight: The amount of liquid dispensed by a weighing pump (in this case, a syringe) during a single dose administration to measure dose delivery.

[0628] Spray pattern (SP): The uniformity of the cross-section of the spray plume at a specified distance (i.e., 30 mm) from the nozzle tip is measured to characterize the equipment performance.

[0629] Spray pattern testing involves capturing a time series of images from a normal view at predetermined distances along the centerline of the spray emitted from the device using laser illumination and a camera. Proveris software is used to automatically analyze the collected image sequences and calculate time-averaged images, which are used to determine the spray pattern profile and other metrics. An automated, non-compacted spray pattern method (combining automated analysis of laser sheet and digital camera imaging) tailored for RSV vaccines or vaccine buffers, based on the Proveris SprayVIEW® instrument platform and in accordance with FDA guidance, was used. All spray pattern metrics were analyzed as follows: Dmax (the longest chord connecting two points on the spray pattern profile and passing through the profile's weighted centroid) Dmin (the shortest chord length connecting two points on the spray pattern profile and passing through the profile's weighted centroid) Ellipticity (the ratio of Dmax to Dmin) Area (the area enclosed by the outline of the spray pattern) A total of 10 devices were measured, covering a total of 20 sprays. The method setup used for spray pattern analysis is detailed in Table 19 below: [Table 19]: Detailed information on spray pattern methods at a distance of 30 mm from the nozzle tip Plume geometry (PG): The geometry (plume width and plume angle) of the spray plume is measured from a side view at a specified distance (i.e., 30 mm) over time to characterize device performance.

[0630] The plume geometry test involves laser illumination and a camera to capture time-series images from a side view along the centerline of the spray emitted from the device. These resulting image sequences provide visualization of the spray and can be analyzed to determine the duration and orientation of the spray and spray particles.

[0631] The Proveris SprayVIEW® instrument platform was used to employ a laser sheet and digital camera-based plume geometry method tailored for RSV vaccines or vaccine buffers. All plume geometry parameters were analyzed according to relevant FDA guidance documents, including the following (2): plume width and plume angle. Plume geometry and metered ejection weight data were collected from a total of 20 ejections across 10 devices. The method setup used for plume geometry is detailed in Table 20 below. Time-averaged plume measurements were analyzed.

[0632] [Table 20]: Detailed information on plume geometry at a distance of 30 mm from the nozzle tip Droplet size distribution (DSD): The size and distribution of atomized droplets are measured on a fully expanded stable phase at a specified distance (i.e., 30 mm) from the nozzle tip using laser diffraction to characterize device performance. The droplet size distribution can be measured on a fully expanded stable phase at a specified distance from the nozzle tip using laser diffraction to measure, for example, Dv10, Dv50, Dv90, span [(Dv90-Dv10) / Dv50], and the percentage of droplets smaller than 10 μm.

[0633] Using the Malvern Spraytec instrument platform, laser diffraction was employed to customize droplet size distributions for RSV vaccines or vaccine buffer solutions. All droplet size distribution (DSD) metrics were analyzed according to the aforementioned FDA guidance, including the following: Dv90: Particle size; 90% of the spray volumetric distribution is below this size. Dv50: Particle size; 50% of the spray volumetric distribution is below this size. Dv10: Particle size; 10% of the spray volumetric distribution is below this size. %V<10 µm: Volume of particles smaller than 10 micrometers Span: Measures the width of the distribution. The narrower the distribution, the smaller the span. The calculation formula is: Span = (Dv90 - Dv10) / Dv50 All samples were tested using a setup that included a 3D dose dispenser and was filled with the nebulizer head.

[0634] Drive mechanism and parameters Given MAD Nasal with a dose dispenser TM The mechanism of the device utilizes Viota ® Force-limited actuation of the unit dose software: When the force limit at the end of the stroke is reached, the software stops actuation, which allows the device to more accurately replicate the human actuation process—the first dose is completed when the plunger strikes the dose dispenser, and the second dose is completed when the plunger strikes the hard stop of the syringe. The following set of actuation parameters (Table 21) was used for all tests. Results and Discussion Comparison of physical properties of test matrices Key physical properties of RSV vaccine and vaccine buffer were compared, and vaccine buffer was used in the analysis to measure dose accuracy, droplet size distribution (DSD), spray plume geometry (PG), and spray pattern (SP). The comparison results are shown in Table 22. The above results demonstrate the similarity of the physical properties of DP and the formulation buffer, with the exception that the buffer matrix has only slightly lower viscosity.

[0635] Test Results A total of 10 vials filled with DP (HD batch #19-035-FP) were tested. Samples were drawn from each vial and then allocated into two tubes (0.1 mL / #1 tube, for the first injection, and 0.1 mL / #2 tube, for the second dose; for example, vial #1 is tubes #1 and #2, vial #2 is tubes #3 and #4, etc.).

[0636] Dosage accuracy calculated by injection weight The dose-weight ratios are summarized in Table 23 below. The raw results were converted to dose-volume using a density value of 1.1377 g / mL. Dosage titration for plaque assay Infectivity titer is a key indicator for formulation and stability evaluation. It is also used as a labeling statement for RSVi dosage (i.e., the high-dose titer target is 6.0 log). 10PFU / dose, where dose = 0.2 mL). The titer reading is directly related to viral activity; a significant decrease in viral activity is considered a decrease in the infectious titer. All titrations were consistent and met the concentration requirements for high-dose RSV vaccines.

[0637] Injection weight The second injection weight test was conducted using an automated actuator with a total of 10 devices (20 sprays). The results are summarized in Table 25. The maximum dose weight of the first spray was 127.60 mg (dose volume 112.07 µL), and the minimum was 108.90 mg (95.72 µL). The average second dose was 106.21 mg (102.11 µL), which was slightly less than the first dose of 121.64 mg (106.92 µL). spray type A total of 10 stops (20 sprays) were tested for spray pattern. The data in Table 24 below shows an average spray area of ​​129.15 mm. 2 The standard deviation was 14.00. The mean ellipticity was 1.25 with a standard deviation of 0.10. The mean Dmax and Dmin were 14.31 mm and 11.47 mm, respectively, with standard deviations of 0.81 and 0.96. Furthermore, compared with the first dose (124.56 mm...), the mean ellipticity was... 2 Compared to the second dose (133.74 mm), 2 On average, the first and second doses showed a slightly larger spray area. Overall, the first and second doses had similar results in terms of spray area, ellipticity, Dmax, and Dmin. Feather geometry (PG) Flow geometry and dose-weight data were collected simultaneously from 10 devices, for a total of 20 injections. As shown in Table 27 below, the flow geometry results were consistent across different devices and between two doses. Droplet size distribution (DSD) The droplet size distribution results were consistent across different devices and between two doses. The results are presented in Table 28. The data below show that the droplet size distribution results were consistent across different devices and between two doses.

[0638] The drug delivery system (DSD) of nasal sprays is an important parameter for nasal products because it significantly affects drug deposition in the nasal cavity. The average droplet size using a nasal nebulizer delivery device is Dv50 = 93.70 μm. The average value of small droplets (%V < 10 μm) is 0.19%, which can be considered safe, and the amount deposited into the lungs is negligible.

[0639] Device stability RSV vaccine formulations and nebulization delivery devices were tested to determine their compatibility at room temperature for up to 24 hours.

[0640] For this simulation study, HD or LD DP vials were thawed at room temperature for 5 to 7 minutes. Device sample preparation followed, with a MAD130 syringe inserted via a rubber stopper into the thawed glass vial, and 300 µL of DP removed after degassing. The nebulizer was then connected, and headspace air was purged by reducing the volume to 200 µL. Each prepared device set was labeled and stored at the corresponding temperatures (i.e., 2°C to 8°C and 25°C) for 2 hours, 4 hours, 6 hours, and 24 hours. Collected samples were frozen at <-60°C and transferred to AnSci for PA testing. Results for infection titer loss are shown below.

[0641] After 24 hours at 5°C ± 3°C, HD (≥ 6.7 log) 10 PFU / mL) and LD (≥ 5.7 log) 10 The average logarithmic decrease / titer loss (PFU / mL) is ≤ 0.1 log 10 PFU / mL.

[0642] After 6 hours at 25°C ± 2°C, HD (≥ 6.7 log) 10 PFU / mL) and LD (≥5.7 log) 10 The average logarithmic decrease / titer loss (PFU / mL) is ≤ 0.1 log 10 PFU / mL; however, after 24 hours at 25°C ± 2°C, its ≥ 0.1 log 10 PFU / mL.

[0643] In summary, the stability data in use described above indicate that for both the HD and LD formulations, the infection titer reached the expected target after incubation at 25°C ± 2°C and 5°C ± 3°C for 6 hours in the MAD130IN device.

[0644] It is recommended to store RSVi HD and LD in the MAD130 IN device at room temperature for up to 6 hours (17).

[0645] Appendix A: Sequence Summary Sequence Description Appendix A provides a list of some of the sequences cited in this article. The amino acid sequences provided are from the N-terminus to the C-terminus. The nucleic acid sequences are from 5' to 3'.

Claims

1. A method for immunizing a pediatric subject against respiratory syncytial virus (RSV) infection, the method comprising administering to the pediatric subject a dose of an nebulized RSV vaccine containing an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L.

2. A method for immunizing a pediatric subject against respiratory syncytial virus (RSV) infection, the method comprising administering a dose of RSV vaccine to the pediatric subject using an intranasal nebulizer delivery device, the RSV vaccine comprising an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L.

3. A method for preventing RSV infection in a pediatric subject or reducing the likelihood of RSV infection in a pediatric subject or preventing or reducing at least one symptom of RSV infection in a pediatric subject, the method comprising administering a dose of an RSV vaccine to the pediatric subject, the RSV vaccine comprising an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L.

4. A method for immunizing a pediatric subject against respiratory syncytial virus (RSV) infection, the method comprising administering to the pediatric subject a dose of an nebulized RSV vaccine containing an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L, wherein the effective amount of the RSV comprises about 5 to about 9 log 10 Plaque-forming units (PFU) / dose, optionally the dose described herein is about 5.6 log. 10 PFU / dosage.

5. A method for immunizing a pediatric subject against respiratory syncytial virus (RSV) infection, the method comprising administering to the pediatric subject a dose of an nebulized RSV vaccine comprising an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L, wherein the effective amount of the RSV comprises approximately 5.4 log 10 PFU / dose, approximately 5.6 log 10 PFU / dose, approximately 6.2 log 10 PFU / dose, approximately 6.4 log 10 PFU / dose or approximately 7.0 log 10 PFU / dosage.

6. A method for immunizing a pediatric subject against respiratory syncytial virus (RSV) infection, the method comprising administering to the pediatric subject a dose of an RSV vaccine comprising an effective amount of live attenuated RSV ∆NS2 / ∆1313 / I1314L, wherein the effective amount of the RSV comprises about 5 to about 9 log 10 Plaque-forming units (PFU) / dose, optionally the dose described herein is about 5.6 log. 10 PFU / dosage.

7. A method for immunizing a pediatric subject against respiratory syncytial virus (RSV) infection, the method comprising administering to the pediatric subject a dose of an RSV vaccine comprising an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L, wherein the effective amount of the RSV comprises approximately 5.4 log 10 PFU / dose, approximately 5.6 log 10 Plaque-forming units (PFU) / dose, approximately 6.2 log 10 PFU / dose, approximately 6.4 log 10 PFU / dose or approximately 7.0 log 10 PFU / dosage.

8. The method of any one of claims 1-3, wherein the effective amount of RSV comprises about 5 log 10 PFU / dose approximately 9 log 10 PFU / dose, optionally approximately 5.4 log 10 PFU / dose, approximately 5.6 log 10 PFU / dose, approximately 6.2 log 10 PFU / dose, approximately 6.4 log 10 PFU / dose or approximately 7.0 log 10 PFU / dosage.

9. The method of any one of claims 1-8, wherein the RSV vaccine is delivered intranasally, and approximately half of the dose is delivered to each nostril of the pediatric subject.

10. The method of claim 9, wherein the dose of the RSV vaccine is delivered in about 0.2 mL, and wherein about 0.1 mL is delivered to each nostril of the pediatric subject.

11. The method of claim 9 or claim 10, wherein the RSV vaccine is delivered sequentially or simultaneously to each nostril of the pediatric subject.

12. The method of any one of claims 1-11, comprising delivering a second dose of the RSV vaccine.

13. The method of claim 12, wherein the second dose comprises about 5.4 log 10 PFU.

14. The method of claim 12, wherein the second dose comprises about 5.6 log 10 PFU.

15. The method of claim 12, wherein the second dose comprises about 6.2 log 10 PFU.

16. The method of claim 12, wherein the second dose comprises about 6.4 log 10 PFU.

17. The method of claim 12, wherein the second dose comprises about 7.0 log 10 PFU.

18. The method of claim 12, wherein the second dose comprises about 5 log 10 PFU to approximately 9 log 10 PFU.

19. The method of any one of claims 12-18, wherein the second dose is administered about 40-50, 45-55, 55-60, 52-60, or 60-65 days after the initial dose.

20. The method of any one of claims 12-19, wherein the second dose is administered at least 56 days after the initial dose.

21. The method of any one of claims 12-20, wherein the second dose is delivered intranasally, and approximately half of the dose is delivered to each nostril of the pediatric subject.

22. The method of any one of claims 12-21, wherein the second dose of the RSV vaccine is delivered in about 0.2 mL, and wherein about 0.1 mL is delivered to each nostril of the pediatric subject.

23. The method of claim 21 or 22, wherein the RSV vaccine is delivered sequentially or simultaneously to each nostril of the pediatric subject.

24. The method of any one of claims 1-23, wherein the pediatric subject is about 6 months to about 22 months old.

25. The method of any one of claims 1-23, wherein the pediatric subject is about 6 months to about 18 months old.

26. The method of any one of claims 1-23, wherein the pediatric subject is at least 6 months old.

27. The method of any one of claims 1-23, wherein the pediatric subject is a full-term newborn.

28. The method of any one of claims 1-23, wherein the pediatric subject is premature.

29. The method of any one of claims 1 and 3-28, wherein the dose of the RSV vaccine is administered to the pediatric subject using an intranasal nebulization delivery device.

30. The method of claim 2 or claim 29, wherein the intranasal nebulization delivery device includes a nozzle to nebulize the RSV vaccine for administration to the pediatric subject.

31. The method of any one of claims 2, 29 or 30, wherein the intranasal nebulization delivery device comprises a tube, a plunger and a dose dispenser.

32. The method of claim 31, further comprising advancing the plunger a first distance within the cylinder to deliver approximately half of the dose of the RSV vaccine into the first nostril of the pediatric subject.

33. The method of claim 31 or 32, further comprising removing the dose dispenser from the plunger.

34. The method of claim 32 or 33, further comprising advancing the plunger a second distance within the cylinder to deliver approximately half of the dose to the second nostril of the pediatric subject.

35. The method of any one of claims 2 or 29-34, wherein the intranasal atomizing delivery device delivers an average droplet size Dv50 of about 10-120 μm.

36. The method of any one of claims 2 or 29-34, wherein the average droplet size D delivered to each nostril of the pediatric subject v50 The range is approximately 10 µm to 120 µm, approximately 30 µm to 120 µm, approximately 50 µm to 110 µm, approximately 70 µm to 110 µm, or approximately 80 µm to 110 µm.

37. The method of any one of claims 2 or 29-34, wherein the intranasal atomizing delivery device delivers an average droplet size Dv50 of at least 30 µm, at least 50 µm, at least 70 µm, at least 80 µm, at least 110 µm, or at least 120 µm.

38. The method of any one of claims 2 or 29-37, wherein the average injection weight delivered to each nostril of the pediatric subject is about 30 mg to about 200 mg, about 50 mg to about 175 mg, about 70 mg to about 160 mg, about 80 mg to about 150 mg, about 95 mg to about 135 mg, about 100 mg to about 130 mg, about 100 mg to about 130 mg, or between about 105 mg and about 130 mg.

39. The method of any one of claims 1-2 or 29-38, wherein the average injection volume delivered to each nostril of the pediatric subject is about 85 µL to about 120 µL, about 90 µL to about 115 µL, or about 95 µL to about 115 µL.

40. The method of any one of claims 1-39, wherein the codon for serine at position 1313 encoding the L protein in the attenuated live RSV is deleted, thereby causing a deletion of amino acid (∆1313) in the L protein.

41. The method according to any one of claims 1-39, wherein the substitution of the amino acid residue of leucine for isoleucine at position 1314 in the attenuated live RSV causes a genetically stable mutation (I1314L) in the L gene.

42. The method of any one of claims 1-41, wherein the attenuated live RSV comprises: Large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N), M2-1 protein, non-structural protein 1 (NS1), glycoprotein (G), fusion protein (F), matrix protein (M), M2-2 protein, and small hydrophobic protein (SH); and A genome or antigenome containing the deletion of a codon encoding serine at position 1313 or the corresponding position of the L protein; a mutation in amino acid sequence residue 1314 or the corresponding position of the L protein, wherein the mutation in amino acid sequence residue 1314 of the L protein is an amino acid substitution of leucine for isoleucine, wherein the leucine is encoded by a codon as shown in CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

43. The method of claim 3, wherein the at least one symptom is selected from the development or onset of upper respiratory tract RSV infection, the development or onset of lower respiratory tract RSV infection, the development or onset of otitis media, the progression of upper respiratory tract RSV infection to lower respiratory tract RSV infection, or the progression to otitis media.

44. The method of claim 3, wherein the at least one symptom is selected from asthma, wheezing, or a combination thereof.

45. The use of an intranasal nebulizer for administering a dose of RSV vaccine to pediatric subjects, the RSV vaccine containing an effective amount of RSV ∆NS2 / ∆1313 / I1314L.

46. ​​The use of an RSV vaccine in the manufacture of a medicine for the prevention of RSV virus infection in pediatric subjects or for reducing the likelihood of RSV virus infection in pediatric subjects, the RSV vaccine comprising an effective amount of RSV ∆NS2 / ∆1313 / I1314L.

47. The use as described in claim 45 or 46, wherein the effective amount of the RSV comprises about 5 log10 PFU to about 9 log10 PFU / dose.

48. The use as claimed in any one of claims 41-43, wherein the effective amount of the RSV comprises about 5.4 log10 PFU, about 5.6 log10 PFU, about 6.2 log10 PFU, about 6.4 log10 PFU, or about 7.0 log10 PFU.

49. The use as described in any one of claims 45-48, wherein the RSV vaccine is delivered intranasally, wherein about half of the dose is delivered to each nostril of the pediatric subject.

50. The use as claimed in claim 49, wherein the dose of the RSV vaccine is delivered in about 0.2 mL, and wherein about 0.1 mL is delivered to each nostril of the pediatric subject.

51. The use as described in claim 49 or 50, wherein the RSV vaccine is delivered sequentially or simultaneously to each nostril of the pediatric subject.

52. The use as described in any one of claims 45-51, comprising delivering a second dose of the RSV vaccine.

53. The use as described in claim 52, wherein the second dose comprises about 5.4 log 10 PFU.

54. The use as described in claim 52, wherein the second dose comprises about 5.6 log 10 PFU.

55. The use as described in claim 52, wherein the second dose comprises about 6.2 log 10 PFU.

56. The use as described in claim 52, wherein the second dose comprises about 6.4 log 10 PFU.

57. The method of claim 52, wherein the second dose comprises about 7.0 log 10 PFU.

58. The use as described in claim 52, wherein the second dose comprises about 5 log 10 PFU to approximately 9 log 10 PFU.

59. The use as claimed in any one of claims 52-58, wherein the second dose is administered about 40-50, 45-55, 55-60, 52-60, or 60-65 days after the initial dose.

60. The use as described in any one of claims 52-58, wherein the second dose is administered at least 56 days after the initial dose.

61. The use as described in any one of claims 52-60, wherein the second dose is delivered intranasally, and approximately half of the dose is delivered to each nostril of the pediatric subject.

62. The use as described in any one of claims 52-61, wherein the second dose of the RSV vaccine is delivered in about 0.2 mL, and wherein about 0.1 mL is delivered to each nostril of the pediatric subject.

63. The use as described in claim 61 or 62, wherein the RSV vaccine is delivered sequentially or simultaneously to each nostril of the pediatric subject.

64. The use as described in any one of claims 45-63, wherein the pediatric subject is about 6 months to about 22 months old.

65. The use as described in any one of claims 45-63, wherein the pediatric subject is about 6 months to about 18 months old.

66. The use as described in any one of claims 45-63, wherein the pediatric subject is at least 6 months old.

67. The use as described in any one of claims 45-66, wherein the pediatric subject is a full-term newborn.

68. The use as described in any one of claims 45-66, wherein the pediatric subject is premature.

69. The use as described in claim 46, wherein the dose of the RSV vaccine is administered to the pediatric subject using an intranasal nebulization delivery device.

70. The use as described in claim 45 or claim 69, wherein the intranasal nebulization delivery device includes a nozzle for nebulizing the RSV vaccine for administration to the pediatric subject.

71. The use as described in any one of claims 45, 69 or 70, wherein the intranasal nebulizer delivery device comprises a tube, a plunger and a dose dispenser.

72. The use as claimed in claim 71, comprising advancing the plunger a first distance within the cylinder to deliver approximately half of the dose of the RSV vaccine into the first nostril of the pediatric subject.

73. The use as described in claim 71 or 72, comprising advancing the plunger a second distance within the cylinder to deliver approximately half of the dose to the second nostril of the pediatric subject.

74. The use as described in any one of claims 45 or 69-73, wherein the intranasal atomizing delivery device delivers an average droplet size D of about 10-120 μm. v50 .

75. The use as described in any one of claims 45 or 69-74, wherein the average droplet size D delivered to each nostril of the pediatric subject v50 The range is approximately 10 µm to 120 µm, approximately 30 µm to 120 µm, approximately 50 µm to 110 µm, approximately 70 µm to 110 µm, or approximately 80 µm to 110 µm.

76. The use as claimed in any one of claims 45 or 69-73, wherein the intranasal atomizing delivery device delivers an average droplet size Dv50 of at least 30 µm, at least 50 µm, at least 70 µm, at least 80 µm, at least 110 µm, or at least 120 µm.

77. The use as claimed in any one of claims 45 or 69-76, wherein the average injection weight delivered to each nostril of the pediatric subject is about 30 mg to about 200 mg, about 50 mg to about 175 mg, about 70 mg to about 160 mg, about 80 mg to about 150 mg, about 95 mg to about 135 mg, about 100 mg to about 130 mg, about 100 mg to about 130 mg, or between about 105 mg and about 130 mg.

78. The use as claimed in any one of claims 45 or 69-77, wherein the average injection volume delivered to each nostril of the pediatric subject is about 85 µL to about 120 µL, about 90 µL to about 115 µL, or about 95 µL to about 115 µL.

79. The use as described in any one of claims 45-78, wherein the codon for serine at position 1313 encoding the L protein in the attenuated live RSV is deleted, thereby causing a deletion of amino acid (∆1313) in the L protein.

80. The use as described in any one of claims 45-79, wherein the substitution of the amino acid residue of leucine for isoleucine at position 1314 in the attenuated active RSV causes a genetically stable mutation (I1314L) in the L gene.

81. The use as described in any one of claims 45-80, wherein the attenuated live RSV comprises: Large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N), M2-1 protein, non-structural protein 1 (NS1), glycoprotein (G), fusion protein (F), matrix protein (M), M2-2 protein, and small hydrophobic protein (SH); and A genome or antigenome containing the deletion of a codon encoding serine at position 1313 or the corresponding position of the L protein; a mutation in amino acid sequence residue 1314 or the corresponding position of the L protein, wherein the mutation in amino acid sequence residue 1314 of the L protein is a leucine-to-isoleucine amino acid substitution, wherein the leucine is encoded by a codon as shown in CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

82. A kit comprising a dose of RSV vaccine and an intranasal nebulizer for administering the RSV vaccine to pediatric subjects, the RSV vaccine comprising an effective amount of attenuated live RSV ∆NS2 / ∆1313 / I1314L.

83. The kit of claim 82, wherein the effective amount of the RSV comprises about 5 log 10 PFU to approximately 9 log 10 PFU / dosage.

84. The kit of claim 82 or claim 83, wherein the effective amount of the RSV comprises about 5.4 log 10 PFU and / or approximately 5.6 log 10 PFU and / or approximately 6.2 log 10 PFU and / or approximately 6.4 log 10 PFU and / or approximately 7.0 log 10 PFU.

85. The kit according to any one of claims 82-84, wherein the kit further comprises a second dose of the RSV vaccine.

86. The kit of claim 85, wherein the second dose comprises an effective amount of the RSV, the effective amount of the RSV comprising about 5 log 10 PFU to approximately 9 log 10 PFU / Second dose.

87. The kit of claim 85, wherein the second dose comprises an effective amount of the RSV, the effective amount of the RSV comprising approximately 5.4 log 10 PFU and / or approximately 5.6 log 10 PFU and / or approximately 6.2 log 10 PFU and / or approximately 6.4 log 10 PFU and / or approximately 7.0 log 10 PFU / Second dose.

88. The kit according to any one of claims 82-87, wherein the first dose and / or the second dose comprises a volume of about 0.2 mL.

89. The kit according to any one of claims 82-88, wherein the intranasal nebulization delivery device includes a nozzle for nebulizing the RSV vaccine.

90. The kit according to any one of claims 82-89, wherein the intranasal nebulization delivery device comprises a tube, a plunger, and a dose dispenser.

91. The kit according to any one of claims 82-90, wherein the intranasal nebulizer delivers an average droplet size D of about 10-120 μm. v50 .

92. The kit according to any one of claims 82-91, wherein the intranasal nebulization delivery device delivers an average droplet size Dv50 of at least 30 µm, at least 50 µm, at least 70 µm, at least 80 µm, at least 110 µm, or at least 120 µm.

93. The kit according to any one of claims 82-92, wherein the intranasal nebulizer delivers an average injection weight of about 30 mg to about 200 mg, about 50 mg to about 175 mg, about 70 mg to about 160 mg, about 80 mg to about 150 mg, about 95 mg to about 135 mg, about 100 mg to about 130 mg, about 100 mg to about 130 mg, or about 105 mg to about 130 mg.

94. The kit according to any one of claims 82-93, wherein the intranasal nebulizer delivers an average injection volume of about 85 µL to about 120 µL, about 90 µL to about 115 µL, or about 95 µL to about 115 µL, which is half the dose.

95. A recombinant infectious respiratory syncytial virus, comprising: Large polymerase protein (L), phosphoprotein (P), nucleocapsid protein (N), M2-1 protein, non-structural protein 1 (NS1), glycoprotein (G), fusion protein (F), matrix protein (M), M2-2 protein, and small hydrophobic protein (SH); and A genome or antigenome containing the deletion of a codon encoding serine at position 1313 or the corresponding position of the L protein; a mutation in amino acid sequence residue 1314 or the corresponding position of the L protein, wherein the mutation in amino acid sequence residue 1314 of the L protein is a leucine-to-isoleucine amino acid substitution, wherein the leucine is encoded by a codon as shown in CTG; a deletion of the NS2 gene; and a nucleotide modification at position 14456 of reference SEQ ID NO: 1, which represents a change from thymine (T) to adenine (A).

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